Polynucleotides encoding novel variants of the TRP channel family member, LTRPC3

ABSTRACT

The present invention provides novel polynucleotides encoding LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

[0001] This application claims benefit to provisional application U.S.Serial No. 60/469,894 filed May 12, 2003; under 35 U.S.C. 119(e). Theentire teachings of the referenced application are incorporated hereinby reference.

FIELD OF THE INVENTION

[0002] The present invention provides novel polynucleotides encodingLTRPC3 polypeptides, fragments and homologues thereof. The presentinvention also provides polynucleotides encoding variants of LTRPC3polypeptides, specifically LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k,and LTRPC3l. Also provided are vectors, host cells, antibodies, andrecombinant and synthetic methods for producing said polypeptides. Theinvention further relates to diagnostic and therapeutic methods forapplying these novel LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, andLTRPC3l polypeptides to the diagnosis, treatment, and/or prevention ofvarious diseases and/or disorders related to these polypeptides. Theinvention further relates to screening methods for identifying agonistsand antagonists of the polynucleotides and polypeptides of the presentinvention.

BACKGROUND OF THE INVENTION

[0003] Intracellular Ca²⁺ plays a pivotal role in various cellfunctions, ranging from exocytosis and contraction to gene expressionand cell differentiation, proliferation and apoptosis. Ca²⁺ entry intocells, particularly in non-excitable cells, can be mediated viastore-operated Ca²⁺ channel (s) (SOC). Following Ca²⁺ release from theintracellular stores, SOC mediate Ca²⁺ influx from the extracellularspace to generate sustained increases in intracellular Ca²⁺concentration and replenish the internal Ca²⁺ stores. The molecularmechanism of SOC activation and the molecular identity of SOC remainselusive. Members of TRP (Transient Receptor Potential) channels, anemerging class of Ca²⁺-permeable cation channel superfamily, are likelycandidates for SOC (reviewed in Trends Neurosci, 23, 159-166, (2000)).

[0004] Human mutations in the genes involved in intracellular Ca²⁺handling result in visual defects, diabetes mellitus, disorders in theskin, skeletal-muscle, nervous, cardiac and vascular systems (reviewedby Missiaen et al., 2000). In addition to the well characterizedvoltage-dependent Ca²⁺ channels, Ca²⁺ pumps and Ca²⁺-permeableligand-gated channels, TRPC (Transient Receptor Potential Channels) isan emerging class of Ca²⁺-permeable cation channel superfamily. All ofthe channels in this family contain a six-trans-membrane domain althoughvarious cellular mechanisms have been implicated in their functions.

[0005] Following the identification of the founding member of thisfamily, DTRP, from the Drosophila mutants trp whose photoreceptorsfailed to generate a sustained receptor potential in response to intensesustained light (Neuron 8, 643-651, (1992)), mammalian homologues havebeen cloned and all of them contain a six-trans-membrane domain followedby a TRP motif (XWKFXR, SEQ ID NO:168), the diagnosed feature of the TRPfamily of proteins. The mutant fly showed a reduced Ca²⁺ selectivity ofthe light response and the channel activity of dTRP depended on PLCactivation was also demonstrated.

[0006] Based on their homology, they are divided into three subfamilies:short (s), osm (o) and long (l). New nomenclature for each subfamily hasrecently been proposed and is as follows: TRPC (canonical), TRPV(vanilloid), and TRPM (melastatin) (Mol. Cell 9, 229-231, (2002)). ThesTRPC subfamily includes TRP1-7. Although the specific physiologicalfunction of each isoform remains to be assigned, it is generallybelieved that they may be involved in Ca²⁺ entry after activation ofreceptors coupling to PLC. The TRP2 is specifically expressed invomeronasal organ and involved in pheromone sensory signaling (Liman, etal., 1999). TRP1 and TRP6 are functioned in vascular smooth muscle cellsand may play a role in controlling smooth muscle tone, arteriosclerosisand neointimal hypoerplasia (Inoue et al., 2001; Xu & Beech, 2001). Ithas been shown that TRP4−/− mice lack an endothelial store-operated Ca²⁺current, which leads to reduced agonist-dependent vasorelaxation(Freichel et al., 2001).

[0007] The first member of oTRPC Subfamily is OSM-9 cloned from C.elegans. It is involved in responses to odorants, high osmotic strength,and mechanical stimulation. Recently, several mammalian homologuesincluding vanilloid receptor (VR1) and vanilloid receptor-like receptor(VRL-1), which may have functions in pain and heat perception (Caterina,1999; Caterina et al., 2000). VR1 has also been shown to be the receptorof anandamide and mediating its vasodilation effect (Zygmunt et al.,1999). OTRPC4 is an osmotically activated channel and a candidateosmoreceptor, may be involved in regulation of cellular volume(Strotmann et al., 2000). CaT1 & ECaC1 may be thecalcium-release-activated calcium channel and involved in Ca²⁺reabsorption in intestine and kidney (Peng, et al, 1999; Yu et al.,2001).

[0008] The function of the lTRPC is less clear. The cloned mammalianlTRPC includes melastatin1/MLSN1/LTRPC1, MTR1/LTRPC5, TRPC7/LTRPC2 andTRP-P8. It is known that melastatin 1 is down regulated in metastaticmelanomas (Duncan et al., 1998) and MTR1 is associated withBeckwith-Wiedemann syndrome and a predisposition to neoplasias (Prawittet al., 2000). TRPC7 is mapped to the chromosome region linked tobipolar affective disorder, nonsyndromic hereditary deafness, Knoblochsyndrome and holosencephaly (Nagamine et al., 1998). TRP-P8 is aprostate-specific gene and up-regulated in prostate cancer and othermalignancies (Tsavaler et al., 2001). A recently clonedTRP-PLIK/hSOC-2/hCRAC-1 exhibits a very interesting feature in that itis a bi-functional protein with kinase and ion channel activities(Runnels et al., 2001). Additionally, a very long TRPC homologue NOMPCwas found in Drosophila and C. elegans. NOMPC was identified as amechanosensitive channel that can detect sound, pressure or movementchanges (Walker et al., 2000).

[0009] Members of the TRPM subfamily are characteristic of theirunusually long cytoplasmic tails at both ends of the channel domain andsome of the family members contain an enzyme domain at the C-terminalregion. Despite their similarities of structure, TRPMs have beenimplicated in a variety of biological functions. TRPM1 is found to bedown-regulated in metastatic melanomas (Cancer Res. 58, 1515-1520,(1998)). TRPM2 is a Ca²⁺-permeable channel that contains an ADP-ribosepyrophosphatase domain and can be activated by ADP-ribose, NAD (Nature411, 595-599, (2001); and Science 293, 1327-1330, (2001)) and changes inredox status (Mol. Cell 9, 163-173, (2002)). TRPM2 is mapped to thechromosome region linked to bipolar affective disorder, nonsyndromichereditary deafness, Knobloch syndrome and holosencephaly (Genomics 54,124-131, (1998)). Two splice variants of TRPM4 have been described.TRPM4a is predominantly a Ca²⁺ permeable channel (Proc. Natl. Acad. Sci.U.S.A. 98, 10692-10697, (2001); whereas TRPM4b conducts monovalentcations upon activation by changes in intracellular Ca²⁺ (Cell 109,397-401, (2002)). TRPM5 is associated with Beckwith-Wiedemann syndromeand a predisposition to neoplasias (Mol. Genet. 9, 203-216, (2001)).TRPM7, another bi-functional protein, has kinase activity in additionalto its ion channel activity. TRPM7 is regulated by Mg²⁺-ATP and/or PIP₂,and required for cell viability (Science 291, 1043-1047, (2001); Nature411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36, (2002)). TRPM8 isup-regulated in prostate cancer and other malignancies (Cancer Res. 61,3760-3769, (2001)). Recently, it has also been shown to be a receptorthat senses cold stimuli (Nature 416, 52-58, (2002); and Cell 108,705-715, (2002)).

[0010] As described herein, the polypeptides of the present inventionare novel variants of the LTRPC3 polypeptide. The LTRPC3 polypeptide isdescribed in co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002;and International Publication No. WO 03/012063, published Feb. 13, 2003;in addition to Co-pending U.S. Ser. No. 10/405,793, filed Mar. 28, 2003)which are hereby incorporated by reference in its entirety.

[0011] Characterization of the LTRPC3 polypeptide led to thedetermination that it is involved in the modulation of the FEN1 DNAbase-excision repair/proliferation modulating protein, either directlyor indirectly.

[0012] In mammalian cells, single-base lesions, such as uracil andabasic sites, appear to be repaired by at least two base excision repair(BER) subpathways: “single-nucleotide BER” requiring DNA synthesis ofjust one nucleotide and “long patch BER” requiring multi-nucleotide DNAsynthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol)accounts for both gap filling DNA synthesis and removal of the5′-deoxyribose phosphate (dRP) of the abasic site, whereas theinvolvement of various DNA polymerases in long patch BER is less wellunderstood.

[0013] Flap endonuclease 1 (Fen1) is a structure-specificmetallonuclease that plays an essential function in DNA replication andDNA repair (Tom, S., Henricksen, L, A., Bambara, R, A, J. Biol, Chem.,275(14):10498-505, (2000)). It interacts like many other proteinsinvolved in DNA metabolic events with proliferating cell nuclear antigen(PCNA), and its enzymatic activity is stimulated by PCNA in vitro by asmuch as 5 to 50 fold (Stucki, M., Jonsson, Z, O., Hubscher, U, J. Biol,Chem., 276(11):7843-9, (2001)). Recently, immunodepletion experiments inhuman lymphoid cell extracts have shown long-patch BER to be dependentupon FEN1 (Prasad, R., Dia, G, L., Bohr, V, A., Wilson, S, H, J. Biol,Chem., 275(6):4460-6, (2000)). In addition, FEN1 has also been shown tocooperate with beta-pol in long patch BER excision and is involved indetermining the predominant excision product seen in cell extracts. Thesubstrate for FEN1 is a flap formed by natural 5′-end displacement ofthe short intermediates of lagging strand replication. FEN1 binds to the5′-end of the flap, tracks to the point of annealing at the base of theflap, and then cleaves the substrate (Tom, S., Henricksen, L, A.,Bambara, R, A, J. Biol, Chem., 275(14): 10498-505, (2000)).

[0014] The FEN1 is also referred to as Rad27. FEN1 plays a critical rolein base-excision repair as evidenced by Saccharomyces cerevisiae FEN1null mutants displaying an enhancement in recombination that increasesas sequence length decreases (Negritto, M, C., Qiu, J., Ratay, D, O.,Shen, B., Bailis, A, M, Mol, Cell, Biol., 21(7):2349-58, (2001)). Thelatter suggests that Rad27 preferentially restricts recombinationbetween short sequences. Since wild-type alleles of both RAD27 and itshuman homologue FEN1 complement the elevated short-sequencerecombination (SSR) phenotype of a rad27-null mutant, this function maybe conserved from yeast to humans. Furthermore, mutant Rad27 and FEN-1enzymes with partial flap endonuclease activity but withoutnick-specific exonuclease activity were shown to partially complementthe SSR phenotype of the rad27-null mutant suggesting that theendonuclease activity of Rad27 (FEN-1) plays a role in limitingrecombination between short sequences in eukaryotic cells. In addition,preliminary data from yeast suggests the FEN-1 deficiencies may resultin genomic instability (Ma, X., Jin, Q., Forsti, A., Hemminki, K., Ku,R, Int, J. Cancer., 88(6):938-42, (2000)). More recently, FEN1 nullmutants results in the expansion of repetitive sequences (Henricksen, L,A., Tom, S., Liu, Y., Bambara, R, A, J. Biol, Chem., 275(22):16420-7,(2000)).

[0015] Aside from the role of FEN1 in base-excision repair, FEN1 hasalso been shown to play a significant role in modulating signaltransduction in proliferating cells. This role is intricately associatedwith the role of FEN1 in DNA replication. Of particular significance isthe observation that FEN1 is a nuclear antigen, that it is expressed bycycling cells, and that it co-localizes with PCNA and polymerase alphaduring S phase. Fen1 expression is topologically regulated in vivo andis associated with proliferative populations (Warbrick, E., Coates, P,J., Hall, P, A, J. Pathol., 186(3):319-24, (1998)). Antibodies have beendescribed by Warbrick et al. that specifically bind FEN1, the assays ofwhich are hereby incorporated herein by reference.

[0016] In addition, experiments in S. cerevisiae using the novelimmunosuppressant agent SR 31747 have shown that SR 31747 arrests cellproliferation by directly targeting sterol isomerase and that FEN1 isrequired to mediate the proliferation arrest induced by ergosteroldepletion (Silve, S., Leplatois, P., Josse, A., Dupuy, P, H., Lanau, C.,Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le, Fur, G.,Caput, D., Ferrara, P., Loison, G, Mol, Cell, Biol., 16(6):2719-27,(1996)).

[0017] Using the above examples, it is clear the availability of a novelcloned transient receptor potential channel family provides anopportunity for adjunct or replacement therapy, and are useful for theidentification of transient receptor potential channel agonists, orstimulators (which might stimulate and/or bias transient receptorpotential channel function), as well as, in the identification oftransient receptor potential channel inhibitors. All of which might betherapeutically useful under different circumstances.

[0018] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, andLTRPC3l polypeptides using recombinant techniques. Synthetic methods forproducing the polypeptides and polynucleotides of the present inventionare provided. Also provided are diagnostic methods for detectingdiseases, disorders, and/or conditions related to the LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, and LTRPC3l polypeptides and polynucleotides,and therapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

[0019] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3g protein having the amino acid sequence shown in FIGS. 1A-E(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone,LTRPC3g (also referred to as TRPM3g, LTRPC3 variant g) deposited as ATCCDeposit Number XXXXX on XXXXX.

[0020] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3h protein having the amino acid sequence shown in FIGS. 2A-F(SEQ ID NO:4) or the amino acid sequence encoded by the cDNA clone,LTRPC3h (also referred to as TRPM3h, and/or LTRPC6 variant h).

[0021] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3i protein having the amino acid sequence shown in FIGS. 3A-F(SEQ ID NO:6) or the amino acid sequence encoded by the cDNA clone,LTRPC3i (also referred to as TRPM3i, and/or LTRPC6 variant i).

[0022] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3j protein having the amino acid sequence shown in FIGS. 4A-F(SEQ ID NO:10) or the amino acid sequence encoded by the cDNA clone,LTRPC3j (also referred to as TRPM3j, and/or LTRPC6 variant j).

[0023] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3k protein having the amino acid sequence shown in FIGS. 5A-F(SEQ ID NO:12) or the amino acid sequence encoded by the cDNA clone,LTRPC3k (also referred to as TRPM3k, and/or LTRPC6 variant k).

[0024] The present invention provides isolated nucleic acid molecules,that comprise, or alternatively consist of, a polynucleotide encodingthe LTRPC3l protein having the amino acid sequence shown in FIGS. 6A-F(SEQ ID NO:14) or the amino acid sequence encoded by the cDNA clone,LTRPC3l (also referred to as TRPM3l, and/or LTRPC6 variant l).

[0025] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells, in addition to their usein the production of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, andLTRPC3l polynucleotides or polypeptides using recombinant techniques.Synthetic methods for producing the polypeptides and polynucleotides ofthe present invention are provided. Also provided are diagnostic methodsfor detecting diseases, disorders, and/or conditions related to theLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and LTRPC3l polypeptidesand polynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

[0026] The invention further provides an isolated LTRPC3g polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0027] The invention further provides an isolated LTRPC3h polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0028] The invention further provides an isolated LTRPC3i polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0029] The invention further provides an isolated LTRPC3j polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0030] The invention further provides an isolated LTRPC3k polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0031] The invention further provides an isolated LTRPC3l polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0032] The invention further relates to a polynucleotide encoding apolypeptide fragment of SEQ ID NO:2, 4, 6, 8, 10 and/or 12, or apolypeptide fragment encoded by the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 3, 5, 7, 9 and/or11.

[0033] The invention further relates to a polynucleotide encoding apolypeptide domain of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 or apolypeptide domain encoded by the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 3, 5, 7, 9 and/or11.

[0034] The invention further relates to a polynucleotide encoding apolypeptide epitope of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 or apolypeptide epitope encoded by the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1, 3, 5, 7, 9 and/or11.

[0035] The invention further relates to a polynucleotide encoding apolypeptide of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 or the cDNA sequenceincluded in the deposited clone, which is hybridizable to SEQ ID NO:1,3, 5, 7, 9 and/or 11, having biological activity.

[0036] The invention further relates to a polynucleotide which is avariant of SEQ ID NO:1, 3, 5, 7, 9 and/or 11.

[0037] The invention further relates to a polynucleotide which is anallelic variant of SEQ ID NO:1, 3, 5, 7, 9 and/or 11.

[0038] The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2, 4, 6, 8, 10 and/or 12.

[0039] The invention further relates to a polynucleotide whichrepresents the complimentary sequence (antisense) of SEQ ID NO:1, 3, 5,7, 9 and/or 11.

[0040] The invention further relates to a polynucleotide capable ofhybridizing under stringent conditions to any one of the polynucleotidesspecified herein, wherein said polynucleotide does not hybridize understringent conditions to a nucleic acid molecule having a nucleotidesequence of only A residues or of only T residues.

[0041] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:2, 4, 6, 8, 10 and/or 12, wherein thepolynucleotide fragment comprises a nucleotide sequence encoding animmunoglobulin protein.

[0042] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:1, 3, or 5 wherein the polynucleotide fragmentcomprises a nucleotide sequence encoding the sequence identified as SEQID NO:2, 4, 6, 8, 10 and/or 12 or the polypeptide encoded by the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1, 3, 5, 7, 9 and/or 11.

[0043] The invention further relates to an isolated nucleic acidmolecule of of SEQ ID NO:1, 3, or 5, wherein the polynucleotide fragmentcomprises the entire nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9and/or 11 or the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1, 3, 5, 7, 9 and/or 11.

[0044] The invention further relates to an isolated nucleic acidmolecule of SEQ ID NO:1, 3, 5, 7, 9 and/or 11, wherein the nucleotidesequence comprises sequential nucleotide deletions from either theC-terminus or the N-terminus.

[0045] The invention further relates to an isolated polypeptidecomprising an amino acid sequence that comprises a polypeptide fragmentof SEQ ID NO:2, 4, 6, 8, 10 and/or 12 or the encoded sequence includedin the deposited clone.

[0046] The invention further relates to a polypeptide fragment of SEQ IDNO:2, 4, 6, 8, 10 and/or 12 or the encoded sequence included in thedeposited clone, having biological activity.

[0047] The invention further relates to a polypeptide domain of SEQ IDNO:2, 4, 6, 8, 10 and/or 12 or the encoded sequence included in thedeposited clone.

[0048] The invention further relates to a polypeptide epitope of SEQ IDNO:2, 4, 6, 8, 10 and/or 12 or the encoded sequence included in thedeposited clone.

[0049] The invention further relates to a full length protein of SEQ IDNO:2, 4, 6, 8, 10 and/or 12 or the encoded sequence included in thedeposited clone.

[0050] The invention further relates to a variant of SEQ ID NO:2, 4, 6,8, 10 and/or 12.

[0051] The invention further relates to an allelic variant of SEQ IDNO:2, 4, 6, 8, 10 and/or 12.

[0052] The invention further relates to a species homologue of SEQ IDNO:2, 4, 6, 8, 10 and/or 12.

[0053] The invention further relates to the isolated polypeptide of ofSEQ ID NO:2, 4, 6, 8, 10 and/or 12, wherein the full length proteincomprises sequential amino acid deletions from either the C-terminus orthe N-terminus.

[0054] The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2, 4, 6, 8, 10and/or 12.

[0055] The invention further relates to a method for preventing,treating, or ameliorating a medical condition, comprising administeringto a mammalian subject a therapeutically effective amount of thepolypeptide of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 or the polynucleotideof SEQ ID NO:1, 3, 5, 7, 9 and/or 11.

[0056] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence orabsence of a mutation in the polynucleotide of SEQ ID NO:1, 3, 5, 7, 9and/or 11; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence orabsence of said mutation.

[0057] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence oramount of expression of the polypeptide of of SEQ ID NO:2, 4, 6, 8, 10and/or 12 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.

[0058] The invention further relates to a method for identifying abinding partner to the polypeptide of SEQ ID NO:2, 4, 6, 8, 10 and/or 12comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2,4, 6, 8, 10 and/or 12 with a binding partner; and (b) determiningwhether the binding partner effects an activity of the polypeptide.

[0059] The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1, 3, 5, 7, 9 and/or 11.

[0060] The invention further relates to a method of identifying anactivity in a biological assay, wherein the method comprises the stepsof (a) expressing SEQ ID NO:1, 3, 5, 7, 9 and/or 11 in a cell, (b)isolating the supernatant; (c) detecting an activity in a biologicalassay; and (d) identifying the protein in the supernatant having theactivity.

[0061] The invention further relates to a process for makingpolynucleotide sequences encoding gene products having altered activityselected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10 and/or 12activity comprising the steps of (a) shuffling a nucleotide sequence ofSEQ ID NO:1, 3, 5, 7, 9 and/or 11, (b) expressing the resulting shufflednucleotide sequences and, (c) selecting for altered activity selectedfrom the group consisting of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 activityas compared to the activity selected from the group consisting of SEQ IDNO:2, 4, 6, 8, 10 and/or 12 activity of the gene product of saidunmodified nucleotide sequence.

[0062] The invention further relates to a shuffled polynucleotidesequence produced by a shuffling process, wherein said shuffled DNAmolecule encodes a gene product having enhanced tolerance to aninhibitor of any one of the activities selected from the groupconsisting of SEQ ID NO:2, 4, 6, 8, 10 and/or 12 activity.

[0063] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, wherein the medical condition isa renal disorder

[0064] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, wherein the medical condition isa neural disorder.

[0065] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, wherein the medical condition isa disorder related to aberrant calcium regulation.

[0066] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, wherein the medical condition isa reproductive disorder.

[0067] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, wherein the medical condition isa neural disorder.

[0068] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a neural disorder.

[0069] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a neural disorder selected from the groupconsisting of: cerebellum disorders, various choroid plexus neoplasms,choroid plexus papilloma, Alzheimer's disease, prion disorders andmultiple sclerosis and movement disorders that involve the cerebellum.

[0070] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a proliferative kidney or renal disorder.

[0071] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is solitary metastasis of the kidney.

[0072] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is renal cell carcinoma.

[0073] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is ovarian cancer or related proliferativecondition of the ovary.

[0074] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is testicular cancer or related proliferativecondition of the testis.

[0075] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is kidney cancer or related proliferativecondition of the kidney.

[0076] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder that maps to or is associated withchromosome locus 9q21.11-21.31.

[0077] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is amyotrophic lateral sclerosis withfrontotemporal dementia, early-onset pulverulent cataract, infantilenephronophthisis, hypomagnesemia with secondary hypocalcemia and/orfamilial hemophagocytic lymphohistiocytosis.

[0078] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0079] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a condition that would benefit from modulationof intracellular Ca++ ion concentrations, Ca++ ion flux, storedintracellular Ca++ ion concentrations, Ca++ ion pump activity, Ca++ ionflow into cell, Ca++ ion flow out of cells, the activation of Ca++senstive proteins, the activation of Ca++ senstive signaling pathways,the activation of kinase-activatible proteins, and the activation ofkinase-dependent signaling pathways.

[0080] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition Ca²+ reabsorption disorders, disorders associatedwith aberrant luminal Ca2+ entry via the epithelial Ca2+ channel (ECaC),disorders associated with aberrant cytosolic diffusion of Ca2+ bound tocalbindin-D28K, disorders associated with aberrant basolateral extrusionof Ca2+ through the Na+/Ca2+ exchanger (NCX), disorders associated withaberrant plasma membrane Ca2+-ATPase (PMCA) activity and/or activation,disorders associated with the hypocalciuric effect of diuretics,disorders associated with the hypocalciuric effect of thiazidediuretics, disorders associated with hormone insufficiencies that affectthe function of the kidney, disorders associated with hormoneinsufficiencies that affect renal Ca2+ homeostasis, disorders associatedwith aberrant renal Ca2+ homeostasis as a result of vitamin Ddeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of PTH deficiency, disorders associated with aberrant renalCa2+ homeostasis as a result of vasopressin deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofprostaglandines deficiency, disorders associated with aberrant renalCa2+ homeostasis as a result of estrogen deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofcalcitonin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of parathyroid hormone deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of atrialnatriuretic peptide deficiency, calcium homeostasis-related disorders,hypercaliuric nephrolithiasis, certain forms of osteoporosis, Gitelman'sdisease, Bartter's syndrom, disorders associated with aberrant functionof kidney tubular epithelium, disorders associated with aberrantfunction of kidney tubular epithelium in the medulla, disordersassociated with aberrant function of kidney tubular epithelium in themedullary rays, disorders associated with aberrant function of kidneytubular epithelium in the periglomerular.

[0081] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition acute kidney failure, chronic kidney failure,cystic kidney disease, horseshoe kidney, hypertensive kidney disease,kidney atrophy, kidney cancer, kidney disease, kidney failure, kidneyinfection, kidney metastasis, kidney stone, nephrolithiasis, multicysticdysplastic kidney disease, and polycystic kidney disease.

[0082] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is DNA-repair deficiencies, particularlybase-excision repair deficiencies, Xeroderma pigmentosum, skin cancer,melanoma, UV senstivity, alkylation sensivity, gamma irradiationsensitivity, pyrimidine dimer sensitivity, chemical mutagenes,lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi'sanemia, ataxia telangiectasia, chromosomal aberrations, blood vesseldilation aberrations in the skin, blood vessel dilation aberrations inthe eye, conditions involving increased levels of apurinic sites,conditions involving increased levels of apyrimidinic sites, conditionsinvolving increased levels of abasic sites, disorders related toaberrant signal transduction, proliferating disorders, and/or cancers.

[0083] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder related to misregulation of FEN1expression and/or activity.

[0084] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, ormodulator thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder that would benefit from increasingmammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0085] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, orantagonists thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder that would benefit from increasingmammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0086] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, oragonists thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder associated with aberrant signaltransduction, proliferating disorders, and/or cancers, particularlyrenal cell carcinomas, testicular cancers, and/or ovarian cancers.

[0087] The invention further relates to a method for preventing,treating, or ameliorating a medical condition with the polypeptide, oragonists thereof, provided as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, inaddition to, its encoding nucleic acid, or a modulator thereof, whereinthe medical condition is a disorder that would benefit from decreasingmammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0088] The invention further relates to a method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject comprising the steps of (a) determining the presence oramount of expression of the polypeptide of of SEQ ID NO:2, 4, 6, 8, 10and/or 12 in a biological sample; (b) and diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide relative to acontrol, wherein said condition is a member of the group consisting ofovarian cancer, testicular cancer, and kidney cancer.

[0089] The present invention is also directed to polynucleotidescomprising at least about 465 nt of the polynucleotide sequence providedas SEQ ID NO:1, 3, 5, 7, 9, and/or 11. Specifically, the presentinvention is directed to a polynucleotide sequence comprisingnucleotides from about nucleotide 72 to about nucleotide 536 of SEQ IDNO:1, 3, 5, 7, 9, and/or 11. In this context “about” includes theparticularly recited value, a value larger or smaller by several (5, 4,3, 2, or 1) nucleotides, at either terminus, or at both termini.

[0090] The present invention is also directed to polypeptides comprisingat least about 155 amino acids of the polypeptides sequence provided asSEQ ID NO:2, 4, 6, 8, 10, and/or 12. Specifically, the present inventionis directed to a polypeptides sequence comprising amino acids from aboutamino acid 1 to about amino acid 155 of SEQ ID NO:2, 4, 6, 8, 10, and/or12. In this context “about” includes the particularly recited value, avalue larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, ateither terminus, or at both termini.

[0091] The invention further relates to a method of identifying acompound that modulates the biological activity of LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l, comprising the steps of, (a)combining a candidate modulator compound with LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, and/or LTRPC3l having the sequence set forth in one ormore of SEQ ID NO:2, 4, 6, 8, 10 and/or 12; and (b) measuring an effectof the candidate modulator compound on the activity of LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l.

[0092] The invention further relates to a method of identifying acompound that modulates the biological activity of a transient receptorpotential channel protein, comprising the steps of, (a) combining acandidate modulator compound with a host cell expressing LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l having the sequenceas set forth in SEQ ID NO:2, 4, 6, 8, 10 and/or 12; and, (b) measuringan effect of the candidate modulator compound on the activity of theexpressed LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l.

[0093] The invention further relates to a method of identifying acompound that modulates the biological activity of LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l, comprising the steps of, (a)combining a candidate modulator compound with a host cell containing avector described herein, wherein LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l is expressed by the cell; and, (b) measuring aneffect of the candidate modulator compound on the activity of theexpressed LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l.

[0094] The invention further relates to a method of screening for acompound that is capable of modulating the biological activity ofLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l, comprisingthe steps of: (a) providing a host cell described herein; (b)determining the biological activity of LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, and/or LTRPC3l in the absence of a modulator compound;(c) contacting the cell with the modulator compound; and (d) determiningthe biological activity of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k,and/or LTRPC3l in the presence of the modulator compound; wherein adifference between the activity of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l in the presence of the modulator compound and inthe absence of the modulator compound indicates a modulating effect ofthe compound.

[0095] The invention further relates to a method of identifying acompound that modulates the biological activity of LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l, comprising the steps of, (a)combining a candidate modulator compound with a host cell containing avector described herein, wherein LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l is expressed by the cell; (b) measuring aneffect of the candidate modulator compound on the activity of theexpressed LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l;wherein step (a) optionally comprises one or more of the followingadditional steps: (i) incubation of said host cells in a medium lackingCa++ ions; (ii) incubation of said host cells in a medium containingCa++ ions; (iii) incubation of said host cells in a medium lacking Ca++ions, followed by addition of Ca++ ion to the medium of said host cellwithin a period either preceding or following incubation of saidcandidate modulator compound; incubation of said host cells underconditions where Ca++ stores within said host cells would be expected tobe at least partially depleted; incubation of said host cells with La³⁺;incubation of said host cells with Gd³⁺; incubation of said host cellswith La³⁺ in a medium lacking Ca++ ions, followed by addition of Ca++ion to the medium of said host cell within a period either preceding orfollowing incubation of said candidate modulator compound; incubation ofsaid host cells with Gd³⁺ in a medium lacking Ca++ ions, followed byaddition of Ca++ ion to the medium of said host cell within a periodeither preceding or following incubation of said candidate modulatorcompound; incubation of said host cells with La³⁺ in a medium containingCa++ ions; incubation of said host cells with Gd³⁺ in a mediumcontaining Ca++ ions; incubation of said host cells with thapsigargin;incubation of said host cells with carbachol; incubation of said hostcells with thapsigargin in a medium lacking Ca++ ions, followed byaddition of Ca++ ion to the medium of said host cell within a periodeither preceding or following incubation of said candidate modulatorcompound; incubation of said host cells with carbachol in a mediumlacking Ca++ ions, followed by addition of Ca++ ion to the medium ofsaid host cell within a period either preceding or following incubationof said candidate modulator compound; incubating said host cells withFluo-4-AM for a defined period, or other fluorescent cytoplasmic Ca++marker amenable to measurement on a FLIPR instrument, such that saidhost cells are loaded with Fluo-4-AM; and/or incubating said host cellswith Fluo-4-AM for a defined period, or other fluorescent cytoplasmicCa++ marker amenable to measurement on a FLIPR instrument (or otherplate reader, preferably a fluorescent plate reader), such that saidhost cells are loaded with Fluo-4-AM, followed by removal ofextracellular Fluo-4-AM.

[0096] The invention further relates to a compound that modulates thebiological activity of human LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l as identified by the methods described herein.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

[0097] The file of this patent contains at least one Figure executed incolor. Copies of this patent with color Figure(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

[0098] FIGS. 1A-E show the polynucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of the novel human transientreceptor potential channel member variant, LTRPC3g, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5201 nucleotides (SEQ ID NO:1), encodinga polypeptide of 1709 amino acids (SEQ ID NO:2). An analysis of theLTRPC3g polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid776 to about amino acid 792 (TM1), from about amino acid 872 to aboutamino acid 888 (TM2), from about amino acid 945 to about amino acid 958(TM3), from about amino acid 972 to about amino acid 989 (TM4), fromabout amino acid 1006 to about amino acid 1023 (TM5), and/or from aboutamino acid 1093 to about amino acid 1113 (TM6) of SEQ ID NO:2represented by double underlining; a predicted TRP domain located fromabout amino acid 1128 to about amino acid 1133 of SEQ ID NO:2represented by light shading; a predicted ion transport signature domainlocated at about amino acid 903 to about amino acid 1114 of SEQ ID NO:2represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1218 to about amino acid 1273 of SEQ ID NO:2represented by italics; and conserved cysteine residues located at aminoacid 249, 364, 420, 574, 672, 737, 754, 976, 1067, 1084, 1097, 1200,1291, and 1552 of SEQ ID NO:2 represented in bold. Conservation ofcysteines at key amino acid residues is indicative of conservedstructural features, which may correlate with conservation of proteinfunction and/or activity.

[0099] FIGS. 2A-F show the polynucleotide sequence (SEQ ID NO:3) anddeduced amino acid sequence (SEQ ID NO:4) of the novel human transientreceptor potential channel member variant, LTRPC3h, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5237 nucleotides (SEQ ID NO:3), encodinga polypeptide of 1721 amino acids (SEQ ID NO:4). An analysis of theLTRPC3h polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid787 to about amino acid 804 (TM1), from about amino acid 884 to aboutamino acid 901 (TM2), from about amino acid 957 to about amino acid 970(TM3), from about amino acid 984 to about amino acid 1001 (TM4), fromabout amino acid 1018 to about amino acid 1035 (TM5), and/or from aboutamino acid 1106 to about amino acid 1125 (TM6) of SEQ ID NO:4represented by double underlining; a predicted TRP domain located fromabout amino acid 1040 to about amino acid 1045 of SEQ ID NO:4represented by light shading; a predicted ion transport signature domainlocated at about amino acid 915 to about amino acid 1126 of SEQ ID NO:4represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1230 to about amino acid 1285 of SEQ ID NO:4represented by italics; and conserved cysteine residues located at aminoacid 249, 364, 420, 586, 749, 766, 988, 1079, 1096, 1109, 1212, 1303,and 1564 of SEQ ID NO:4 represented in bold. Conservation of cysteinesat key amino acid residues is indicative of conserved structuralfeatures, which may correlate with conservation of protein functionand/or activity.

[0100] FIGS. 3A-F show the polynucleotide sequence (SEQ ID NO:5) anddeduced amino acid sequence (SEQ ID NO:6) of the novel human transientreceptor potential channel member variant, LTRPC3i, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5237 nucleotides (SEQ ID NO:5), encodinga polypeptide of 1721 amino acids (SEQ ID NO:6). An analysis of theLTRPC3i polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid775 to about amino acid 792 (TM1), from about amino acid 872 to aboutamino acid 888 (TM2), from about amino acid 945 to about amino acid 958(TM3), from about amino acid 972 to about amino acid 988 (TM4), fromabout amino acid 1006 to about amino acid 1023 (TM5), and/or from aboutamino acid 1115 to about amino acid 1125 (TM6) of SEQ ID NO:6represented by double underlining; a predicted TRP domain located fromabout amino acid 1140 to about amino acid 1145 of SEQ ID NO:6represented by light shading; a predicted ion transport signature domainlocated at about amino acid 915 to about amino acid 1126 of SEQ ID NO:6represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1230 to about amino acid 1285 of SEQ ID NO:6represented by italics; and conserved cysteine residues located at aminoacid 249, 364, 420, 574, 672, 754, 976, 1212, 1303, and 1564 of SEQ IDNO:6 represented in bold. Conservation of cysteines at key amino acidresidues is indicative of conserved structural features, which maycorrelate with conservation of protein function and/or activity.

[0101] FIGS. 4A-F show the polynucleotide sequence (SEQ ID NO:7) anddeduced amino acid sequence (SEQ ID NO:8) of the novel human transientreceptor potential channel member variant, LTRPC3j, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5171 nucleotides (SEQ ID NO:7), encodinga polypeptide of 1699 amino acids (SEQ ID NO:8). An analysis of theLTRPC3j polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid765 to about amino acid 782 (TM1), from about amino acid 862 to aboutamino acid 879 (TM2), from about amino acid 935 to about amino acid 948(TM3), from about amino acid 962 to about amino acid 979 (TM4), fromabout amino acid 996 to about amino acid 1013 (TM5), and/or from aboutamino acid 1083 to about amino acid 1103 (TM6) of SEQ ID NO:8represented by double underlining; a predicted TRP domain located fromabout amino acid 1118 to about amino acid 1123 of SEQ ID NO:8represented by light shading; a predicted ion transport signature domainlocated at about amino acid 893 to about amino acid 1104 of SEQ ID NO:8represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1208 to about amino acid 1263 of SEQ ID NO:8represented by italics; and conserved cysteine residues located at aminoacid 249, 364, 420, 574, 662, 727, 744, 966, 1057, 1074, 1087, 1190,1281, and 1547 of SEQ ID NO:8 represented in bold. Conservation ofcysteines at key amino acid residues is indicative of conservedstructural features, which may correlate with conservation of proteinfunction and/or activity.

[0102] FIGS. 5A-F show the polynucleotide sequence (SEQ ID NO:9) anddeduced amino acid sequence (SEQ ID NO:10) of the novel human transientreceptor potential channel member variant, LTRPC3k, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5207 nucleotides (SEQ ID NO:9), encodinga polypeptide of 1711 amino acids (SEQ ID NO:10). An analysis of theLTRPC3e polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid777 to about amino acid 794 (TM1), from about amino acid 874 to aboutamino acid 891 (TM2), from about amino acid 947 to about amino acid 960(TM3), from about amino acid 974 to about amino acid 991 (TM4), fromabout amino acid 1008 to about amino acid 1025 (TM5), and/or from aboutamino acid 1095 to about amino acid 1115 (TM6) of SEQ ID NO:10represented by double underlining; a predicted TRP domain located fromabout amino acid 1130 to about amino acid 1135 of SEQ ID NO:10represented by light shading; a predicted ion transport signature domainlocated at about amino acid 905 to about amino acid 1116 of SEQ ID NO:10represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1220 to about amino acid 1275 of SEQ ID NO:10represented by italics; and conserved cysteine residues located at aminoacid 249, 364, 420, 586, 674, 739, 756, 978, 1069, 1086, 1099, 1202,1293, and 1559 of SEQ ID NO:10 represented in bold. Conservation ofcysteines at key amino acid residues is indicative of conservedstructural features, which may correlate with conservation of proteinfunction and/or activity.

[0103] FIGS. 6A-F show the polynucleotide sequence (SEQ ID NO:11) anddeduced amino acid sequence (SEQ ID NO:12) of the novel human transientreceptor potential channel member variant, LTRPC3l, of the presentinvention. The standard one-letter abbreviation for amino acids is usedto illustrate the deduced amino acid sequence. The polynucleotidesequence contains a sequence of 5276 nucleotides (SEQ ID NO:1), encodinga polypeptide of 1734 amino acids (SEQ ID NO:12). An analysis of theLTRPC3l polypeptide determined that it comprised the following features:six transmembrane domains (TM1 thru TM6) located from about amino acid800 to about amino acid 817 (TM1), from about amino acid 897 to aboutamino acid 914 (TM2), from about amino acid 970 to about amino acid 983(TM3), from about amino acid 997 to about amino acid 1014 (TM4), fromabout amino acid 1031 to about amino acid 1048 (TM5), and/or from aboutamino acid 1118 to about amino acid 1137 (TM6) of SEQ ID NO:12represented by double underlining; a predicted TRP domain located fromabout amino acid 1153 to about amino acid 1158 of SEQ ID NO:12represented by light shading; a predicted ion transport signature domainlocated at about amino acid 928 to about amino acid 1136 of SEQ ID NO:12represented by dark shading; a predicted coiled-coil domain located atabout amino acid 1243 to about amino acid 1298 of SEQ ID NO:12represented by italics; and conserved cysteine residues located at aminoacid 249, 389, 599, 697, 762, 779, 1001, 1092, 1109, 1122, 1225, 1316,and 1582 of SEQ ID NO:12 represented in bold. Conservation of cysteinesat key amino acid residues is indicative of conserved structuralfeatures, which may correlate with conservation of protein functionand/or activity.

[0104] FIGS. 7A-G show the regions of identity and similarity betweenthe LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and LTRPC3lpolypeptides of the present invention to another member of humantransient receptor potential channel family, specifically, the humanmelastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQID NO:13). The alignment was created using the CLUSTALW algorithmdescribed elsewhere herein using default parameters (CLUSTALWparameters: gap opening penalty: 10; gap extension penalty: 0.5; gapseparation penalty range: 8; percent identity for alignment delay: 40%;and transition, weighting: 0). The darkly shaded amino acids representregions of matching identity. The lightly shaded amino acids representregions of matching similarity. Dots between residues indicate gappedregions for the aligned polypeptides.

[0105]FIG. 8 shows an expression profile of a variant of the humantransient receptor potential channel family members of the presentinvention, LTRPC3 (Co-pending U.S. Ser. No. 10/210,152, filed Aug. 1,2002; International Publication No. WO 03/012063, published Feb. 13,2003; SEQ ID NO:15). The figure illustrates the relative expressionlevel of LTRPC3 amongst various mRNA tissue sources. As shown,transcripts corresponding to LTRPC3 expressed predominately in kidneytissue. The LTRPC3 polypeptide was also expressed significantly inspinal cord, testis, and brain. Expression data was obtained bymeasuring the steady state LTRPC3 mRNA levels by RT-PCR using the PCRprimer pair provided as SEQ ID NO: 375 and 376 as described herein.

[0106]FIG. 9 shows an expression profile of a variant of the humantransient receptor potential channel family members of the presentinvention, LTRPC3 (Co-pending U.S. Ser. No. 10/210,152, filed Aug. 1,2002; International Publication No. WO 03/012063, published Feb. 13,2003; SEQ ID NO:15). The figure illustrates the relative expressionlevel of LTRPC3 amongst various mRNA tissue, and cell sources. As shown,transcripts corresponding to LTRPC3 expressed predominately in kidneytissue. The LTRPC3 polypeptide was also expressed significantly inbrain, and testis. Expression data was obtained by probing a Northernblot using a LTRPC3 645-bp PCR amplified fragment from SEQ ID NO:14 asdescribed herein.

[0107]FIG. 10 shows a table illustrating the percent identity andpercent similarity between the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and LTRPC3l polypeptides of the present invention with thehuman melastatin 1 protein (Melastatin1; Genbank Accession No.gi|3243075; SEQ ID NO:13). The percent identity and percent similarityvalues were determined based upon the GAP algorithm (GCG suite ofprograms; and Henikoff, S. and Henikoff, J. G., Proc. Natl. Acad. Sci.USA 89: 10915-10919(1992)).

[0108] FIGS. 11A-I shows the regions of identity between the LTRPC3g(SEQ ID NO:2), LTRPC3h (SEQ ID NO:4), LTRPC3i (SEQ ID NO:6), LTRPC3j(SEQ ID NO:8), LTRPC3k (SEQ ID NO:10), and LTRPC3l (SEQ ID NO:12)polypeptides of the present invention to the LTRPC3 variant (SEQ IDNO:15), in addition to splice variants of LTRPC3, including LTRPC3b (SEQID NO:16), LTRPC3c (SEQ ID NO:17), LTRPC3d (SEQ ID NO:18), LTRPC3e (SEQID NO:19), and LTRPC3f (SEQ ID NO:20). The alignment was created usingthe CLUSTALW algorithm described elsewhere herein using defaultparameters (CLUSTALW parameters: gap opening penalty: 10; gap extensionpenalty: 0.5; gap separation penalty range: 8; percent identity foralignment delay: 40%; and transition, weighting: 0). The darkly shadedamino acids represent regions of matching identity. The lightly shadedamino acids represent regions of matching similarity. Dots betweenresidues indicate gapped regions for the aligned polypeptides.

[0109]FIG. 12 shows an expanded expression profile of a variant of thehuman transient receptor potential channel family members of the presentinvention, LTRPC3 (Co-pending U.S. Ser. No. 10/210,152, filed Aug. 1,2002; International Publication No. WO 03/012063, published Feb. 13,2003; SEQ ID NO:15; in addition to Co-pending U.S. Ser. No. 10/405,793,filed Mar. 28, 2003). The figure illustrates the relative expressionlevel of LTRPC3 amongst various mRNA tissue sources. As shown, theLTRPC3 polypeptide was expressed predominately in the brain,specifically the cerebellum, choroid plexus, the locus coeruleus, theposterior hypothalamus and the substantia nigra. Expression of LTRPC3was also significantly expressed in the kidney, with higher levelsobserved in the cortex than in the medulla or pelvis. LTRPC3 was alsosignificantly expressed in the spinal cord, testis, and to a lesserextent in other tissues as shown. Expression data was obtained bymeasuring the steady state LTRPC3 mRNA levels by quantitative PCR usingthe PCR primer pair provided as SEQ ID NO:306 and 307, and Taqman probe(SEQ ID NO:308) as described in Example 4 herein.

[0110]FIG. 13 shows an expanded expression profile of a variant of thehuman transient receptor potential channel family members of the presentinvention, LTRPC3 (Co-pending U.S. Ser. No. 10/210,152, filed Aug. 1,2002; International Publication No. WO 03/012063, published Feb. 13,2003; SEQ ID NO:15; which is hereby incorporated by reference herein intheir entirety). The figure illustrates the relative expression level ofLTRPC3 amongst various mRNA tissue sources isolated from normal andtumor tissues. As shown, the LTRPC3 polypeptide was differentiallyexpressed in renal, testicular, and ovarian cancers compared to eachrespective normal tissue. Expression data was obtained by measuring thesteady state LTRPC3 mRNA levels by quantitative PCR using the PCR primerpair provided as SEQ ID NO:306 and 307, and Taqman probe (SEQ ID NO:308)as described in Example 4 herein.

[0111]FIG. 14 shows a confocal-image of demonstrating that the LTRPC3variant is expressed as an integral membrane protein. The left top panelshows the image obtained with a Fluorescein filter; the left bottompanel shows the image obtained with a DAPI filter; the right top panelshows the image obtained using transmitted light; and the right bottompanel is the merged picture of the above three images. One of cellsexpressing LTRPC3 on the plasma membrane regions is indicated by anarrow. Forty-eight hours after transfection, LTRPC3 expressing HEK 293cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton,and labeled with fluorescein conjugated anti-HA (3F10) 48 hours posttransfection. The cell nuclei were stained with DAPI. Microscopy wasperformed as described in Example 6 herein.

[0112]FIG. 15 shows that the LTRPC3 variant mediatesconcentration-dependent permeability to Ca²⁺. Transfected HEK 293 cellsloaded with Fluo-4 were incubated in 1 mM Ca²⁺ solution (A), or anominally Ca²⁺-free solution (B), and 0, 1, 3 and 10 mM Ca²⁺ solutionswere added respectively to cells as indicated. The left panels representvector-transfected control cells, and the right panels representLTRPC3-transfected cells. Physiometric methods were performed asdescribed in Example 7 herein.

[0113]FIG. 16 shows Ca²⁺ entry currents are induced upon store depletionin LTRPC3 variant expressing HEK 293 cells. 2 mM thapsigargin (A), or 50μM carbachol (B) was first added to LTRPC3 variant expressing HEK 293cells in the absence of external Ca²⁺. After 9 minutes, 2 mM Ca²⁺solution was added to the cell medium at the indicated times. Currentsfor vector transfected HEK 293 cells, and LTRPC3 variant expressing HEK293 cells are labeled accordingly. Panel (C) shows LTRPC3 variantmediated Ca²⁺ currents are inhibited by La³⁺ and Gd³⁺. Cells loaded withFluo-4 were incubated in 1 mM Ca²⁺ solution and treated with 100 μM ofLa³⁺ (“w/ La³⁺”), Gd³⁺ (“w/ Gd³⁺”) or without blockers (“w/o blocker”)for 10 min prior to the addition of 10 mM Ca²⁺ to the cell medium asindicated. Curves represent averages of three independent experiments,each involving at least 8 wells per condition. Physiometric methods wereperformed as described in Example 7 herein.

[0114]FIG. 17 shows In situ Hybridization (ISH) results of the LTRPC3variant (see co-pending U.S. Ser. No. 10/405,793, filed Mar. 28, 2003;which is hereby incorporated by reference herein in its entirety).LTRPC3 mRNA expression was localized in the collecting tubularepithelium of human kidney medulla, medullary rays, and periglomerularregions, with the highest expression localized in medulary tubules.Bright field (i and iv) and dark field (ii and v) photomicrographs showthe hybridization signal from antisense hTRMP3 riboprobe as aggregatesof fine granules in the cytoplasm of the collecting tubular epithelialcells (arrows) in longitudinal (i and ii) and transverse (iv and v)sections. Low background signal is shown by the control LTRPC3 senseriboprobe in photographs iii and vi (the collecting tubules areindicated by arrowheads). Scale bar denotes 50 μm. ISH methods wereperformed as described in Example 8 herein.

[0115]FIG. 18 shows an expanded confocal-image demonstrating that theLTRPC3 variant is expressed as an integral membrane protein (seeco-pending U.S. Ser. No. 10/405,793, filed Mar. 28, 2003; which ishereby incorporated by reference herein in its entirety). The left toppanel (i) shows the image obtained with anti-HA staining (green) whichis specific for expressed HA-tagged LTRPC3; the right top panel (ii)shows the image obtained with CM-DiI staining (red), a lipophilic cellmembrane marker; the right bottom panel (iii) shows a merged imageshowing anti-HA, CM-DiI, and DAPI (nuclear stain, blue), withco-localization of LTRPC3 and CM-DiI appearing as yellow; and the leftbottom panel (iv) showing the intensity profiles for anti-HA staining(green) and CM-DiI staining (red) along the path indicated by the whiteline in panel iii, showing overlap of anti-HA staining with CM-DiIstaining in a quantitative manner. Scale bar denotes 10 μm. Microscopywas performed as described in Example 9 herein.

[0116]FIG. 19 shows that LTRPC3 variant (“TRPM3”)-mediated Ca²⁺ entrycan be inhibited by Gd³⁺ (see co-pending U.S. Ser. No. 10/405,793, filedMar. 28, 2003; which is hereby incorporated by reference herein in itsentirety). Panels A, C and E show results obtained forvector-transfected cells, while Panels B, D and F show results obtainedfor TRPM3-transfected cells. Panels A and B, show TRPM3-mediatedconcentration-dependent Ca²⁺ entry was partially inhibited by Gd³⁺.Cells loaded with Fluo-4 were incubated in 1 mM Ca²⁺ solution andtreated with 100 μM of Gd³⁺ (red) or without blockers (black) for 10 minprior to the addition of 10 mM Ca²⁺ to the bath solution as indicated.Panels C-F, show that both Tg (Panels C and D) and CCh (Panels E and F)induced LTRPC3 variant-mediated Ca²⁺ entry can be inhibited by Gd³⁺.Cells loaded with Fluo-4 were incubated in a nominally Ca²⁺-free mediaand treated without blockers (black) or with 100 μM of Gd³⁺ (red) for 10min prior to the addition of 2 μM TG (Panels C and D, first arrow) or 50μM CCh (Panels E and F, first arrow), Ca²⁺ entry was stimulated by thebath addition of 10 mM Ca²⁺ (second arrow). Representative traces fromone of three independent experiments are shown; each trace is the meanof 12 wells per condition. Physiometric methods were performed asdescribed in Example 10 herein.

[0117]FIG. 20 shows a schematic representation of the genomic cloneAL136545 that was found to contain three new exons that extend the 5′upstream region of the LTRPC3 gene. As shown, the AL136545 clone alsocontains exons 4, 5, part of exon 6, in addition to a significantportion containing intronic sequence. The three exons of clone AL136545were used to extend the polynucleotide sequence of the LTRPC3 clones,and variants thereof, to arrive at the LTRPC3g-l variant polynucleotidesequences of the present invention.

[0118]FIG. 21 shows a schematic representation of the extendedN-terminal portion of the polypeptide sequence of the LTRPC3g protein ofthe present invention (SEQ ID NO:2). Protein domain analysis of theextended N-terminal portion of the LTRPC3g polypeptide using HMMSEARCHagainst the Pfam protein domain database indicated that it contained oneNSF domain and one Nucleoporin_FG domain. NSF domains have bee proposedto function in the regulation of intracellular traffic via fusion of twolipid bilayers (Baldini et al.). The presence of this domain isconsistent with the data provided in FIGS. 14 and 18 that demonstratethat the LTRPC3 variant is a membrane protein, in addition to the dataprovided in FIGS. 15, 16, and 19 which demonstrate that the LTRPC3variant suggest the LTRPC3 variant has storage-operated Ca²⁺ channelactivity. Nucleoporin_FG repeats are found in diverse nucleoporins andmay confer function consistent with Ca²⁺ channel activity.

[0119] Table I provides a summary of the novel polypeptides and theirencoding polynucleotides of the present invention.

[0120] Table II illustrates the preferred hybridization conditions forthe polynucleotides of the present invention. Other hybridizationconditions may be known in the art or are described elsewhere herein.

[0121] Table III provides a summary of various conservativesubstitutions encompassed by the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0122] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. The LTRPC3 variants ofthe present invention represent variants of the LTRPC3 polypeptide(Co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002; InternationalPublication No. WO 03/012063, published Feb. 13, 2003; SEQ ID NO:15).Likewise, all references to “LTRPC3” shall be construed to apply to“LTRPC3”, “LTRPC3g”, “LTRPC3h”, “LTRPC3i”, “LTRPC3j”, “LTRPC3k”, and/or“LTRPC3l” unless otherwise specified herein.

[0123] The invention provides a novel human sequences that encode novelhuman transient receptor potential channel family members that arevariants of the LTRPC3 polypeptide. Specifically, the present inventionprovides polynucleotide sequences that encode LTRPC3 variants LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and LTRPC3l, which all belong to thelTRPC subfamily. Each of the LTRPC3 variants of the present inventionshares significant homology with other transient receptor potentialchannel family members, such as human melatonin receptor 1. Transcriptsfor the LTRPC3 variant were found predominately in kidney, spinal cord,testis, and brain suggesting that the invention potentially modulatesleukocyte proliferation, differentiation, migration, and activation inthese tissues. Since the polynucleotides of the present inventionrepresent what is believed to be the full-length variants of the LTRPC3polynucleotide, the polynucleotide variants of the present inventionhave been tentatively named LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k,and LTRPC3l, which are distinguished from the LTRPC3 splice variantsLTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, and LTRPC3f which are also disclosedin co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002.

[0124] In the present invention, “isolated” refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring), and thus is altered “by the hand of man” from itsnatural state. For example, an isolated polynucleotide could be part ofa vector or a composition of matter, or could be contained within acell, and still be “isolated” because that vector, composition ofmatter, or particular cell is not the original environment of thepolynucleotide. The term “isolated” does not refer to genomic or cDNAlibraries, whole cell total or mRNA preparations, genomic DNApreparations (including those separated by electrophoresis andtransferred onto blots), sheared whole cell genomic DNA preparations orother compositions where the art demonstrates no distinguishing featuresof the polynucleotide/sequences of the present invention.

[0125] In specific embodiments, the polynucleotides of the invention areat least 15, at least 30, at least 50, at least 100, at least 125, atleast 155, at least 500, or at least 1000 continuous nucleotides but areless than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment,polynucleotides of the invention comprise a portion of the codingsequences, as disclosed herein, but do not comprise all or a portion ofany intron. In another embodiment, the polynucleotides comprising codingsequences do not contain coding sequences of a genomic flanking gene(i.e., 5′ or 3′ to the gene of interest in the genome). In otherembodiments, the polynucleotides of the invention do not contain thecoding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,4, 3, 2, or 1 genomic flanking gene(s).

[0126] As used herein, a “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or the cDNA containedwithin the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without a signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

[0127] In the present invention, the full length sequence identified asSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQID NO:11 was often generated by overlapping sequences contained inmultiple clones (contig analysis). A representative clone containing allor most of the sequence for SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11 was deposited with the American TypeCulture Collection (“ATCC”). As shown in Table 1, each clone isidentified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure. The deposited clone isinserted in the XXXXX plasmid (Strategene) as described herein.

[0128] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequnencer (such as the Model 373, preferably a Model 3700, fromApplied Biosystems, Inc.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were pridcted by translationof a DNA sequence determined above. Therefore, as is known in the artfor any DNA seuqnece detemrined by this automated approach, anynucleotide seqence determined herein may contain some errors. Nucleotidesequences determined by automation are typically at least about 90%identical, more typically at least about 95% to at least about 99.9%identical to the actual nucleotide seqnece of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart. As is also known in the art, a single insertion or deletion in adetemrined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded bt the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

[0129] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A-E (SEQ ID NO:1), a nucleic acid molecule of thepresent invention encoding the LTRPC3g polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A-E (SEQ ID NO:1) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0130] Using the information provided herein, such as the nucleotidesequence in FIGS. 2A-F (SEQ ID NO:3), a nucleic acid molecule of thepresent invention encoding the LTRPC3h polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 2A-F (SEQ ID NO:3) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0131] Using the information provided herein, such as the nucleotidesequence in FIGS. 3A-F (SEQ ID NO:5), a nucleic acid molecule of thepresent invention encoding the LTRPC3i polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 3A-F (SEQ ID NO:5) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0132] Using the information provided herein, such as the nucleotidesequence in FIGS. 4A-F (SEQ ID NO:7), a nucleic acid molecule of thepresent invention encoding the LTRPC3j polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 4A-F (SEQ ID NO:7) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0133] Using the information provided herein, such as the nucleotidesequence in FIGS. 5A-F (SEQ ID NO:9), a nucleic acid molecule of thepresent invention encoding the LTRPC3k polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 5A-F (SEQ ID NO:7) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0134] Using the information provided herein, such as the nucleotidesequence in FIGS. 6A-F (SEQ ID NO:11), a nucleic acid molecule of thepresent invention encoding the LTRPC3l polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 6A-F (SEQ ID NO:11) wasdiscovered through the application of bioinformatic methods to humangenomic and EST sequences.

[0135] A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, the complements thereof,to polynucleotide sequences encoding the sequences contained in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, the complements thereof, orthe cDNA within the clone deposited with the ATCC. “Stringenthybridization conditions” refers to an overnight incubation at 42 degreeC. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65 degreeC.

[0136] Also contemplated are nucleic acid molecules that hybridize tothe polynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

[0137] Note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

[0138] Of course, a polynucleotide which hybridizes only to polyA+sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in thesequence listing), or to a complementary stretch of T (or U) residues,would not be included in the definition of “polynucleotide,” since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

[0139] The polynucleotide of the present invention can be composed ofany polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, the polynucleotidecan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

[0140] The polypeptide of the present invention can be composed of aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann N Y Acad Sci 663:48-62 (1992).)

[0141] As will be appreciated by the skilled practitioner, should theamino acid fragment comprise an antigenic epitope, for example,biological function per se need not be maintained. The terms LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l, polypeptide andLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l protein areused interchangeably herein to refer to the encoded product of theLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l nucleic acidsequence according to the present invention.

[0142] “SEQ ID NO:1”, “SEQ ID NO:3”, “SEQ ID NO:5”, “SEQ ID NO:7”, “SEQID NO:9”, and “SEQ ID NO:11” refer to polynucleotide sequences, while“SEQ ID NO:2”, “SEQ ID NO:4”, “SEQ ID NO:6”, “SEQ ID NO:8”, “SEQ IDNO:10”, and “SEQ ID NO:12” refer to polypeptide sequences, all sequencesbeing identified by an integer specified in Table 1 herein.

[0143] “A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

[0144] The term “organism” as referred to herein is meant to encompassany organism referenced herein, though preferably to eukaryoticorganisms, more preferably to mammals, and most preferably to humans.

[0145] As used herein the terms “modulate or modulates” refer to anincrease or decrease in the amount, quality or effect of a particularactivity, DNA, RNA, or protein.

[0146] It is another aspect of the present invention to providemodulators of the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/orLTRPC3l protein and LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/orLTRPC3l peptide targets which can affect the function or activity ofLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l in a cell inwhich LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3lfunction or activity is to be modulated or affected. In addition,modulators of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/orLTRPC3l can affect downstream systems and molecules that are regulatedby, or which interact with, LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k,and/or LTRPC3l in the cell. Modulators of LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, and/or LTRPC3l include compounds, materials, agents,drugs, and the like, that antagonize, inhibit, reduce, block, suppress,diminish, decrease, or eliminate LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l function and/or activity. Such compounds,materials, agents, drugs and the like can be collectively termed“antagonists”. Alternatively, modulators of LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, and/or LTRPC3l include compounds, materials, agents,drugs, and the like, that agonize, enhance, increase, augment, oramplify LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3lfunction in a cell. Such compounds, materials, agents, drugs and thelike can be collectively termed “agonists”.

[0147] The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that discribed by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).

[0148] The polynucleotide and polypeptides of the present invention areuseful as probes for the identification and isolation of full-lengthcDNAs and/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarays.

[0149] In addition, polynucleotides and polypeptides of the presentinvention may comprise one, two, three, four, five, six, seven, eight,or more membrane domains.

[0150] Also, in preferred embodiments the present invention providesmethods for further refining the biological function of thepolynucleotides and/or polypeptides of the present invention.

[0151] Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

[0152] In preferred embodiments, the invention provides methods foridentifying the glycosylation sites inherent in the polynucleotides andpolypeptides of the invention, and the subsequent alteration, deletion,and/or addition of said sites for a number of desirable characteristicswhich include, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

[0153] In further preferred embodiments, methods are provided forevolving the polynucleotides and polypeptides of the present inventionusing molecular evolution techniques in an effort to create and identifynovel variants with desired structural, functional, and/or physicalcharacteristics.

[0154] The present invention further provides for other experimentalmethods and procedures currently available to derive functionalassignments. These procedures include but are not limited to spotting ofclones on arrays, micro-array technology, PCR based methods (e.g.,quantitative PCR), anti-sense methodology, gene knockout experiments,and other procedures that could use sequence information from clones tobuild a primer or a hybrid partner.

Polynucleotides and Polypeptides of the Invention

[0155] Features of the Polypeptide Encoded by Gene No:1

[0156] The polypeptide of this gene provided as SEQ ID NO:2 (FIGS.1A-E), encoded by the polynucleotide sequence according to SEQ ID NO:1(FIGS. 1A-E), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3g, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3gpolypeptide with this protein is provided in FIGS. 7A-G.

[0157] The LTRPC3g polypeptide was determined to share 65.7% identityand 73.5% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0158] The LTRPC3g protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0159] Specifically, the LTRPC3g (SEQ ID NO:2) polypeptide represents anovel variant of the LTRPC3 (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:15) polypeptide. The LTRPC3g represents a novelmember of the TRPM subfamily.

[0160] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0161] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art. Moreover, since the LTRPC3gpolypeptide represents a variant of the LTRPC3 polypeptide, it isexpected that it will share similar biological activity with LTRPC3,some of which is described herein.

[0162] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3g polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 1A-E. The transmembrane domains arelocated from about amino acid 776 to about amino acid 792 (TM1), fromabout amino acid 872 to about amino acid 888 (TM2), from about aminoacid 945 to about amino acid 958 (TM3), from about amino acid 972 toabout amino acid 989 (TM4), from about amino acid 1006 to about aminoacid 1023 (TM5), and/or from about amino acid 1093 to about amino acid1113 to about amino acid 1062 (TM6) of SEQ ID NO:2. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0163] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:22), IVKFWFYTLAYIGYLMLF (SEQ ID NO:23),VTDLIAILLFSVGM (SEQ ID NO:24), RVIYCVNIIYWYIRLLDI (SEQ ID NO:25),MMIDMMYFVIIMLVVLMS (SEQ ID NO:26), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:27). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3gtransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0164] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3g, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3g polypeptide,particularly for the development of antibodies specific to theseregions.

[0165] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:28),NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:29),ILRLQDQPFRSDG (SEQ ID NO:30), FGVNKYLGPYVMMIGK (SEQ ID NO:31), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP(SEQ ID NO:32). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the LTRPC3ginter-transmembrane polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0166] The LTRPC3g polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 364, 420, 574, 672, 737, 754,976, 1067, 1084, 1097, 1200, 1291, and 1552 of SEQ ID No: 2 (FIGS.1A-E). Conservation of cysteines at key amino acid residues isindicative of conserved structural features, which may correlate withconservation of protein function and/or activity.

[0167] In confirmation of the LTRPC3g representing a member of thetransient receptor channel family, the LTRPC3g polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1128 to about amino acid 1133 8 of SEQ ID NO:2. In this context,the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0168] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:33).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3g TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0169] In further confirmation of the LTRPC3g representing a member ofthe transient receptor channel family, the LTRPC3g polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 903 to about amino acid 1114 of SEQ ID NO:2.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0170] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:34).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3g ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0171] The LTRPC3g polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1218 to aboutamino acid 1273 of SEQ ID NO:2. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0172] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERLT (SEQ IDNO:35). Polynucleotides encoding this polypeptide are also provided. Thepresent invention also encompasses the use of this LTRPC3g coiled-coildomain polypeptide as an immunogenic and/or antigenic epitope asdescribed elsewhere herein.

[0173] LTRPC3g polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3g by identifying mutations in the LTRPC3g gene using LTRPC3gsequences as probes or by determining LTRPC3g protein or mRNA expressionlevels. LTRPC3g polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3g peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3g.

[0174] Consistent with LTRPC3g representing a transient receptorpotential channel, immunohistochemical experiments have shown that theLTRPC3 variant (SEQ ID NO:15) localizes to the cell membrane (see FIG.17 and Example 6). Specifically, the complete open reading frame ofLTRPC3 with a C-terminal HA tag was transiently transfected into HEK 293cells to assess its cellular localization. The HA-tagged LTRPC3 wasdetected using a fluorescein-conjugated anti-HA antibody and a laserscanning confocal microscope which produces a green fluorescent signal.The green fluorescent signal was exclusively detected at the subplasmamembrane region of the transfected cells (see FIG. 17), which isconsistent with LTRPC3 being an integral membrane protein. Under theseconditions approximately ˜70% of cells are expressing LTRPC3. Theexpression of full-length protein was accessed with immunoblot using ananti-HA antibody and detected as the expected size of ˜170 kD (data notshown).

[0175] Since the LTRPC3g polypeptide represents an N-terminally extendedvariant of the LTRPC3 polypeptide (see polypeptide alignment provided inFIG. 11A-I), LTRPC3g is expected to have the same or similar function asthe LTRPC3 polypeptide. Moreover, the LTRPC3g polypeptide is expected tolocalize to the cell membrane.

[0176] Additional characterization for the LTRPC3 variant protein isprovided in Co-pending U.S. Ser. No. 10/405,793, filed Mar. 28, 2003;which is hereby incorporated by reference herein in its entirety).

[0177] Additionally, anti-HA staining was found to be associated withthe membrane marker CM-DiI, indicating LTRPC3 protein in or near theplasmalemmal compartment of transfected cells (see FIG. 21).Plasmalemmal localization is consistent with the function of the TRPfamily as Ca²⁺-permeable cation channels. LTRPC3 was also observed inintracellular compartments, possibly resulting from overexpression inthis heterologous expression system, as observed with other ion channels(Marshall, J., et al., (1995) Neuron 14, 211-215.).

[0178] The LTRPC3g polypeptide is expected to also localize in or nearthe plasmalemmal compartment.

[0179] Moreover, physiometric studies have shown that LTRPC3 is afunctional Ca²⁺ permeable channel (see FIGS. 18 and 19, and Example 7).LTRPC3g function was assessed using a Fluorometric Imaging Plate Reader(FLIPR ) that measures real-time intracellular fluorescence changes.Cells transiently transfected with vector or LTRPC3-HA were loaded withthe cytoplasmic Ca²⁺ indicator Fluro-4 in a 1 M Ca²⁺ solution. Additionof Ca²⁺ to the media resulted in a concentration-dependent influx ofCa²⁺ into LTRPC3-expressing cells (FIG. 18; right panels), indicatingthat LTRPC3 is a functional Ca²⁺ channel. In contrast,vector-transfected cells showed minimal Ca²⁺ influx under the sameexperimental conditions (FIG. 18, left panels). The non-transfectedcells were indistinguishable from the vector-transfected cells (data notshown). Therefore, LTRPC3 is a constitutively active channel capable ofmediating Ca²⁺ influx.

[0180] The LTRPC3g polypeptide is also expected to be a constitutivelyactive channel capable of mediating Ca²⁺ influx.

[0181] To further address the mechanism of LTRPC3-mediated Ca²⁺ entry,similar Ca²⁺ addition experiments were performed on transfected cellsincubated (˜30 min) in a nominally Ca²⁺-free solution. Previous studieshave shown that lowering extracellular Ca²⁺ concentration belowphysiological levels can deplete intracellular Ca²⁺ stores in many celltypes including HEK 293 (EMBO J. 17, 4274-4282, (1998)). Incubatingvector-transfected HEK 293 cells in a nominally Ca²⁺-free solution gaverise to Ca²⁺ entry that was dependent on the concentration of Ca²⁺ addedto the buffers, indicating Ca²⁺ influx was mediated through endogenousSOCs in HEK293 cells (FIG. 18, left panels). In LTRPC3 cells, the Ca²⁺transients triggered by similar Ca²⁺ treatment were much larger (FIG.18, right panels). This Ca²⁺ entry observed in LTRPC3 cells incubated inCa²⁺-free media were greater than those observed in 1 mM Ca²⁺ media,indicating that LTRPC3-mediated Ca²⁺ entry can be potentiated by thestore-depletion.

[0182] The LTRPC3g polypeptide is also expected to mediate Ca²⁺ entryand that LTRPC3g-mediated Ca²⁺ entry is expected to be potentiated bystore-depletion.

[0183] The store-operated mechanism of LTRPC3-mediated Ca²⁺ influx wastested further by passively depleting Ca²⁺ stores with thapsigargin(TG), an inhibitor of microsomal Ca²⁺ ATPases that pumps ions from thecytosol back into the stores. Addition of 2 μM thapsigargin equivalentlydepleted Ca²⁺ stores in LTRPC3-HA- and vector-transfected cells (FIG.19A). Following store depletion with TG, addition of Ca²⁺ to the bufferinduced a much larger Ca²⁺ entry in LTRPC3 cells compared to the vectorcontrol cells.

[0184] Receptor-mediated Ca²⁺ entry was also more pronounced inLTRPC3-HA-transfected cells. Carbachol (CCh) can activate an endogenousmuscarinic receptor and trigger IP3 production, leading tostore-depletion in HEK 293 cells. The addition of 50 μM of CCh caused atransient and rapid intracellular Ca²⁺ increase in both LTRPC3- andvector-transfected cells (FIG. 19B). After the store depletion with CCh,adding of Ca²⁺ to the buffer induced a much larger influx of Ca²⁺ intoLTRPC3 cells, as compared to vector control cells. These results showthat after store depletion with TG or CCh LTRPC3-transfected cellsexhibit an increased Ca²⁺ influx when compared to control cells.

[0185] The lanthanides, gadolinium (Gd³⁺) and lanthanum (La³⁺), arenoneselective Ca²⁺-permeable channel blockers, often used as part of thecharacterization of overexpressed TRP channels. Both lanthanides blockedLTRPC3 Ca²⁺ conductance, although La³⁺ was more potent (FIG. 19C). Inthe presence of 1 mM Ca²⁺ in which endogenous SOCs is minimallyactivated (FIG. 18A), pre-treatment with 100 μM of La³⁺ and Gd³⁺ blockedLTRPC3 Ca²⁺ currents, stimulated by adding 10 mM Ca²⁺, by 67 and 39%,respectively. These results indicated that LTRPC3 mediated currents arenot non-specific leak currents resulting from protein overexpression.

[0186] Additional experiments were performed to further confirm the Ca²⁺store-depleted activation of LTRPC3 using Gd³⁺. The effects of 100 μMGd³⁺ on Ca²⁺ permeability were tested in vector- and LTRPC3-transfectedcells. The minimal Ca²⁺ influx observed upon addition of 10 mM Ca²⁺ tothe cells (cells were incubated in the presence of 1 mM Ca²⁺) invector-transfected cells (see FIG. 22A) was strongly inhibited by 100 μMGd³⁺. In contrast, 100 μM Gd³⁺ inhibited Ca²⁺ entry, induced by adding10 mM Ca²⁺, by 53% in LTRPC3-transfected cells (see FIG. 22B). Gd³⁺reduced fluorescence units in vector-transfected cells from 1470±140 to−58±8 and in LTRPC3-transfected cells from 6000±322 to 2080±199 (n=12).Fluorescence values were measured 150 seconds after adding 10 mM Ca²⁺and percent blockade was calculated as 1 minus F_(LTRPC3)−F_(vector) inthe presence of Gd³⁺ divided by F_(LTRPC3)−F_(vector) without blocker.The effects of Gd³⁺ on LTRPC3-mediated Ca²⁺ entry, induced by 10 mMCa²⁺, in the presence of thapsigargin or carbachol was also examined.Cells were incubated in nominally Ca²⁺-free medium for thapsigargin andcarbachol experiments. Gd³⁺ inhibited Ca²⁺ entry by 51% after depletionof intracellular stores with thapsigargin (see FIG. 22B). Gd³⁺ reducedpeak fluorescence after 10 mM Ca²⁺ addition in vector-transfected cellsfrom 26444±2410 to 1316±60 and in LTRPC3-transfected cells from37676±2425 to 6783±250 (see FIGS. 22C and D, respectively; n=12). Gd³⁺inhibited Ca²⁺ entry by 72% after depletion of intracellular stores withcarbachol. Gd³⁺ reduced peak Ca²⁺ fluorescence in vector-transfectedcells from 9327±466 to 453±15 and in LTRPC3-transfected cells from14747±988 to 1975±79 (see FIGS. 22E and F, respectively; n=12). Theseresults show that, under identical conditions, the endogenous Ca²⁺ entrypathway was strongly blocked by application of 100 μM Gd³⁺ whereas theLTRPC3-mediated pathway was partially blocked (53%, see FIGS. 22A andB). Stimulation of Ca²⁺ entry in LTRPC3-transfected cells in thepresence of thapsigargin or carbachol was also partially blocked by 100μM Gd³⁺. These results are consistent with the hypothesis that LTRPC3mediates a Ca²⁺ entry pathway that apparently is distinct from theendogenous Ca²⁺ entry pathways present in HEK 293 cells.

[0187] The LTRPC3g polypeptide is also expected to mediate a Ca²⁺ entrypathway that is distinct from the endogenous Ca²⁺ entry pathways presentin HEK 293 cells.

[0188] LTRPC3 is constitutively active but can be potentiated bystore-depletion and is partially sensitive to La³⁺ and Gd³⁺ blockade.LTRPC3 is believed to represent the first member of the TRPM subfamilythat exhibits this store-operated mechanism, although some members ofTRPC subfamily have been considered for this role. TRPM1 and TRPM4a areconstitutive Ca²⁺ permeable channels but it is unclear whether they canbe stimulated by store-depletion (Proc. Natl. Acad. Sci. U.S.A. 98,10692-10697, (2001)). Distinct from TRPM4a, TRPM4b is directly activatedby changes in intracellular Ca²⁺ without significant permeation of Ca²⁺(Cell 109, 397-401, (2002)). TRPM2 is activated by ADP-ribose, NAD andchanges in redox status (Nature 411, 595-599, (2001); Science 293,1327-1330, (2001); and Mol. Cell 9, 163-173, (2002)). TRPM7 is regulatedby Mg²⁺-ATP and/or PIP2 (Science 291, 1043-1047, (2001); Nature 411,690-695, (2001); and Nat. Cell Biol. 4, 329-36 (2002)). TRPM8 isactivated by cold temperatures and cooling agents (Nature 416, 52-58,(2002); and Cell 108, 705-715, (2002)). Therefore, in conjunction withits fairly restricted tissue expression, which is not observed with anyother family members, LTRPC3 may have a unique biological function inhumans.

[0189] Expression profiling designed to measure the steady state mRNAlevels encoding the LTRPC3 polypeptide showed predominately highexpression levels in kidney. The LTRPC3 polypeptide was alsosignificantly expressed in spinal cord, testis, and brain (as shown inFIG. 6).

[0190] Moreover, Northern hybridizations of the LTRPC3 mRNA confirmedthe predominately high expression levels in kidney, and significantexpression levels in testis, and brain (as shown in FIG. 7). TheNorthern hybridization was not performed on spinal cord tissue.

[0191] Expanded analysis of LTRPC3 expression levels by TaqMan™quantitative PCR (see FIG. 12) confirmed that the LTRPC3 polypeptide isexpressed in kidney, brain, testis (FIGS. 6 and 7), although higherexpression levels were observed in brain than previously appreciated.LTRPC3 mRNA was expressed predominately in the brain, specifically thecerebellum, choroid plexus, the locus coeruleus, the posteriorhypothalamus and the substantia nigra. Expression of LTRPC3 was alsosignificantly expressed in the kidney, with higher levels observed inthe cortex than in the medulla or pelvis. LTRPC3 was also significantlyexpressed in the spinal cord, testis, and to a lesser extent in othertissues as shown.

[0192] The LTRPC3g polypeptide of the present invention is expected toshare the same or similar expression profile as that of the LTRPC3polypeptide (SEQ ID NO:15).

[0193] Therefore, LTRPC3g polynucleotides and polypeptides, includingmodulators or fragments thereof, may be useful in treating, diagnosing,prognosing, and/or preventing a variety of neurological conditions, inparticular various choroid plexus neoplasms, choroid plexus papilloma,Alzheimer's disease, prion disorders and multiple sclerosis and movementdisorders that involve the cerebellum. Based upon the expression patternof the LTRPC3 variant in kidney, the novel TRP family member LTRPC3gvariant may also be the cause of solitary metastasis in the choroidplexus, a rare type of carcinoma. For example, it has been shown thatout of 15 cases of solitary metastasis of the choroid plexus, fiveoriginated from renal cell carcinoma (Neurol. Med. Chir. (Tokyo) 1997December;37(12):916-9). Additionally, given the rather selectiveexpression of the LTRPC3 variant in the choroid plexus and renaltissues, it may be possible that altered function of LTRPC3g may beresponsible for solitary metastasis and renal carcinoma. LTRPC3gpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing a variety of renal disorders, particularly solitarymetastasis in the choroid plexus, and renal carcinoma.

[0194] Morever, an additional analysis of LTRPC3 expression levels byTaqMan™ quantitative PCR (see FIG. 13) in disease cells and tissuesindicated that the LTRPC3 polypeptide is differentially expressed inrenal, testicular, and ovarian tumor tissues. In the renal tumor tissueresults, an average of 2 out of 3 matched samples, which represent 3 outof 4 samples total, showed a significant decrease in LTRPC3 steady stateRNA levels in tumor compared to control samples. In the testicular tumortissue results, differential expression of LTRPC3 in testicular cancerswas observed with all 5 tumor samples showing a significant reduction insteady-state RNA levels compared to two control samples. In the ovariantumor tissue results, differential expression of LTRPC3 in ovariancancers was observed with 3 tumor samples showing a significantreduction in steady-state RNA levels compared to five control samples.

[0195] The LTRPC3g polypeptide is expected to share the same or similardifferential expression patterns observed for the LTRPC3 variant.

[0196] Thus, loss of LTRPC3g expression during tumor progression mightcontribute to the metastatic process by altering internal calcium storesin a manner that reflects a loss of cellular control on apoptosis.Restoring LTRPC3g function might provide a novel therapeutic approach totreating certain cancers. Therefore, LTRPC3g polynucleotides andpolypeptides, including modulators or fragments thereof, particularlyagonists of LTRPC3g activity or expression, may be useful in treating,diagnosing, prognosing, ameloriating, and/or preventing a variety ofcancers and proliferative conditions, particularly of the kidney,testis, and ovaries.

[0197] An additional analysis of LTRPC3 localized expression wasassessed using In situ hybridization techniques (ISH). LTRPC3 expressionwas localized to the cytoplasm of collecting tubular epithelium in themedulla, medullary rays, and periglomerular regions (see FIG. 20, Platesi, ii, iv and v). Tubules in the medulla exhibited the most intenseexpression. Other tubular epithelia, e.g., proximal convoluted tubularepithelium, exhibited minimal expression. Expression patterns werecompared to LTRPC3 sense mRNA-labeled human kidney sections as negativecontrols (FIG. 20, Plates iii and vi) and to human lysozyme antisensemRNA labeled human kidney sections as positive controls (data notshown).

[0198] The observed for the LTRPC3 variant mRNA expression in humankidney, suggests that LTRPC3g may play a role in renal handling ofcalcium and other cations. The kidney plays a major role in Ca²⁺homeostasis. LTRPC3g could be involved in Ca²⁺ absorption directly dueto its Ca²⁺ permeability. Indeed, the in situ hybridization analysisdescribed herein demonstrates that LTRPC3 is predominantly expressed inthe connecting tubule, which has frequently been implicated in activetranscellular Ca²⁺ reabsorption (see Hoenderop, J. G., et al., (2002)Annu. Rev. Physiol. 64, 529-549; which is hereby incorporated byreference herein in its entirety). Alternatively, LTRPC3g may functionas an SOC that regulates Ca²⁺ absorption. In the kidney, Ca²⁺ absorptionis regulated by agonists such as calcitonin, parathyroid hormone, andparathyroid hormone-related peptide through their respective Gprotein-coupled receptors, and downstream SOCs (Friedman, P. A., et al.,(1995) Physiol. Rev. 75, 429-471; which is hereby incorporated byreference herein in its entirety).

[0199] Potentially, LTRPC3g may be involved in the pathogenesis ofcalcium homeostasis-related disorders such as hypercaliuricnephrolithiasis, certain forms of osteoporosis, Gitelman's disease andBartter's syndrom.

[0200] In preferred embodiments, LTRPC3g polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0201] In preferred embodiments, LTRPC3g polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0202] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0203] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0204] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1 modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3g to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3g activity or a compound that increased LTRPC3g message levelswould be a desired invention for cancer therapy.

[0205] In preferred embodiments, LTRPC3g polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0206] Moreover, LTRPC3g polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0207] In preferred embodiments, antagonists directed against LTRPC3gare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0208] Moreover, antagonists directed against LTRPC3g are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0209] In preferred embodiments, agonists directed against LTRPC3g areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0210] Moreover, agonists directed against LTRPC3g are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0211] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3gpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0212] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0213] Thus, the LTRPC3g polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0214] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3gpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0215] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3gpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0216] In preferred embodiments, LTRPC3g polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3gpolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0217] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3g polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0218] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0219] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0220] Thus, the LTRPC3g polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0221] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis. signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0222] In preferred embodiments, LTRPC3g polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0223] In more preferred embodiments, LTRPC3g polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0224] LTRPC3g polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3gpolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0225] The LTRPC3g gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3g can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0226] Moreover, given the selective expression in kidney, LTRPC3g couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3g chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3g is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3g inthe disease. Therefore, it is possible that LTRPC3g may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0227] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3g may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3g may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0228] In addition, LTRPC3g polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0229] The LTRPC3g polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3g polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0230] In addition, antagonists of the LTRPC3g polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0231] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3g polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3g, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0232] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3g gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0233] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3g, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3g and assessing their ability to grow wouldprovide convincing evidence the LTRPC3g polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0234] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0235] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0236] In the case of LTRPC3g transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0237] In preferred embodiments, the following N-terminal LTRPC3gdeletion polypeptides are encompassed by the present invention:M1-T1709, G2-T1709, K3-T1709, K4-T1709, W5-T1709, R6-T1709, D7-T1709,A8-T1709, A9-T1709, E10-T1709, M11-T1709, E12-T1709, R13-T1709,G14-T1709, C15-T1709, S16-T1709, D17-T1709, R18-T1709, E19-T1709,D20-T1709, N21-T1709, A22-T1709, E23-T1709, S24-T1709, R25-T1709,R26-T1709, R27-T1709, S28-T1709, R29-T1709, S30-T1709, A31-T1709,S32-T1709, R33-T1709, G34-T1709, R35-T1709, F36-T1709, A37-T1709,E38-T1709, S39-T1709, W40-T1709, K41-T1709, R42-T1709, L43-T1709,S44-T1709, S45-T1709, K46-T1709, Q47-T1709, G48-T1709, S49-T1709,T50-T1709, K51-T1709, R52-T1709, S53-T1709, G54-T1709, L55-T1709,P56-T1709, S57-T1709, Q58-T1709, Q59-T1709, T60-T1709, P61-T1709,A62-T1709, Q63-T1709, K64-T1709, S65-T1709, W66-T1709, I67-T1709,E68-T1709, R69-T1709, A70-T1709, F71-T1709, Y72-T1709, K73-T1709,R74-T1709, E75-T1709, C76-T1709, V77-T1709, H78-T1709, I79-T1709,I80-T1709, P81-T1709, S82-T1709, T83-T1709, K84-T1709, D85-T1709,P86-T1709, H87-T1709, R88-T1709, C89-T1709, C90-T1709, C91-T1709,G92-T1709, R93-T1709, L94-T1709, I95-T1709, G96-T1709, Q97-T1709,H98-T1709, V99-T1709, G100-T1709, L10-T1709, T102-T1709, P103-T1709,S104-T1709, I105-T1709, S106-T1709, V107-T1709, L108-T1709, Q109-T1709,N110-T1709, E111-T1709, K112-T1709, N113-T1709, E114-T1709, S115-T1709,R116-T1709, L117-T1709, S118-T1709, R119-T1709, N120-T1709, D121-T1709,I122-T1709, Q123-T1709, S124-T1709, E125-T1709, K126-T1709, W127-T1709,S128-T1709, I129-T1709, S130-T1709, K131-T1709, H132-T1709, T133-T1709,Q134-T1709, L135-T1709, S136-T1709, P137-T1709, T138-T1709, D139-T1709,A140-T1709, F141-T1709, G142-T1709, T143-T1709, I144-T1709, E145-T1709,F146-T1709, Q147-T1709, G148-T1709, G149-T1709, G150-T1709, H151-T1709,S152-T1709, N153-T1709, K154-T1709, and/or A155-T1709 of SEQ ID NO:2.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3g deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0238] In preferred embodiments, the following C-terminal LTRPC3gdeletion polypeptides are encompassed by the present invention:M1-T1709, M1-H1708, M1-K1707, M1-S1706, M1-E1705, M1-F1704, M1-S1703,M1-Q1702, M1-F1701, M1-A1700, M1-S1699, M1-T1698, M1-R1697, M1-S1696,M1-L1695, M1-R1694, M1-R1693, M1-M1692, M1-S1691, M1-L1690, M1-S1689,M1-D1688, M1-G1687, M1-R1686, M1-G1685, M1-E1684, M1-P1683, M1-K1682,M1-S1681, M1-S1680, M1-K1679, M1-S1678, M1-R1677, M1-Q1676, M1-F1675,M1-P1674, M1-N1673, M1-Q1672, M1-L1671, M1-S1670, M1-A1669, M1-T1668,M1-N1667, M1-R1666, M1-Q1665, M1-R1664, M1-D1663, M1-L1662, M1-K1661,M1-D1660, M1-S1659, M1-I1658, M1-S1657, M1-F1656, M1-S1655, M1-K1654,M1-R1653, M1-T1652, M1-H1651, M1-A1650, M1-Y1649, M1-P1648, M1-A1647,M1-S1646, M1-P1645, M1-E1644, M1-E1643, M1-A1642, M1-S1641, M1-Y1640,M1-S1639, M1-N1638, M1-A1637, M1-R1636, M1-E1635, M1-I1634, M1-K1633,M1-P1632, M1-V1631, M1-T1630, M1-I1629, M1-N1628, M1-N1627, M1-S1626,M1-L1625, M1-T1624, M1-R1623, M1-E1622, M1-S1621, M1-N1620, M1-D1619,M1-G1618, M1-E1617, M1-Q1616, M1-S1615, M1-S1614, M1-I1613, M1-A1612,M1-I1611, M1-T1610, M1-A1609, M1-R1608, M1-R1607, M1-G1606, M1-K1605,M1-A1604, M1-E1603, M1-N1602, M1-E1601, M1-E1600, M1-S1599, M1-D1598,M1-S1597, M1-S1596, M1-P1595, M1-H1594, M1-S1593, M1-L1592, M1-E1591,M1-A1590, M1-E1589, M1-R1588, M1-E1587, M1-P1586, M1-H1585, M1-C1584,M1-C1583, M1-T1582, M1-L1581, M1-D1580, M1-E1579, M1-V1578, M1-K1577,M1-D1576, M1-G1575, M1-L1574, M1-G1573, M1-G1572, M1-P1571, M1-F1570,M1-A1569, M1-A1568, M1-R1567, M1-D1566, M1-A1565, M1-I1564, M1-A1563,M1-Q1562, M1-P1561, M1-A1560, M1-N1559, M1-V1558, M1-C1557, M1-R1556,M1-T1555, M1-D1554, M1-I1553, M1-C1552, M1-D1551, M1-T1550, M1-I1549,M1-S1548, M1-T1547, M1-Y1546, M1-E1545, M1-A1544, M1-T1543, M1-K1542,M1-V1541, M1-P1540, M1-V1539, M1-G1538, M1-F1537, M1-N1536, M1-A1535,M1-Y1534, M1-Y1533, M1-S1532, M1-R1531, M1-S1530, M1-P1529, M1-S1528,M1-F1527, M1-M1526, M1-F1525, M1-S1524, M1-H1523, M1-S1522, M1-K1521,M1-V1520, M1-I1519, M1-P1518, M1-A1517, M1-E1516, M1-E1515, M1-L1514,M1-L1513, M1-F1512, M1-P1511, M1-T1510, M1-T1509, M1-A1508, M1-L1507,M1-Y1506, M1-R1505, M1-S1504, M1-S1503, M1-K1502, M1-S1501, M1-R1500,M1-E1499, M1-I1498, M1-T1497, M1-H1496, M1-Y1495, M1-M1494, M1-P1493,M1-P1492, M1-E1491, M1-S1490, M1-D1489, M1-W1488, M1-P1487, M1-N1486,M1-Q1485, M1-C1484, M1-E1483, M1-P1482, M1-L1481, M1-H1480, M1-T1479,M1-Y1478, M1-D1477, M1-S1476, M1-S1475, M1-F1474, M1-S1473, M1-R1472,M1-T1471, M1-D1470, M1-M1469, M1-S1468, M1-T1467, M1-I1466, M1-D1465,M1-E1464, M1-F1463, M1-D1462, M1-I1461, M1-S1460, M1-R1459, M1-S1458,M1-P1457, M1-P1456, M1-R1455, M1-D1454, M1-T1453, M1-P1452, M1-A1451,M1-L1450, M1-T1449, M1-A1448, M1-Y1447, M1-A1446, M1-S1445, M1-S1444,M1-S1443, M1-P1442, M1-A1441, M1-T1440, M1-S1439, M1-P1438, M1-V1437,M1-P1436, M1-T1435, M1-S1434, M1-F1433, M1-S1432, M1-P1431, M1-E1430,M1-G1429, M1-L1428, M1-G1427, M1-L1426, M1-I1425, M1-N1424, M1-V1423,M1-S1422, M1-N1421, 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M1-E684,M1-S683, M1-A682, M1-E681, M1-H680, M1-A679, M1-M678, M1-A677, M1-K676,M1-C675, M1-L674, M1-K673, M1-C672, M1-A671, M1-V670, M1-L669, M1-A668,M1-K667, M1-A666, M1-M665, M1-A664, M1-E663, M1-E662, M1-G661, M1-H660,M1-Q659, M1-W658, M1-F657, M1-F656, M1-L655, M1-A654, M1-M653, M1-K652,M1-Q651, M1-R650, M1-K649, M1-M648, M1-L647, M1-V646, M1-A645, M1-W644,M1-V643, M1-M642, M1-L641, M1-E640, M1-H639, M1-F638, M1-P637, M1-F636,M1-P635, M1-F634, M1-H633, M1-N632, M1-I631, M1-E630, M1-P629, M1-D628,M1-D627, M1-L626, M1-D625, M1-I624, M1-D623, M1-V622, M1-E621, M1-E620,M1-E619, M1-R618, M1-K617, M1-K616, M1-T615, M1-T614, M1-K613, M1-R612,M1-G611, M1-R610, M1-R609, M1-L608, M1-P607, M1-I606, M1-D605, M1-D604,M1-E603, M1-M602, M1-G601, M1-L600, M1-L599, M1-K598, M1-L597, M1-A596,M1-K595, M1-P594, M1-R593, M1-K592, M1-P591, M1-G590, M1-F589, M1-L588,M1-N587, M1-H586, M1-Y585, M1-L584, M1-T583, M1-R582, M1-F581, M1-R580,M1-K579, M1-R578, M1-T577, M1-Y576, M1-N575, M1-C574, M1-R573, M1-Y572,M1-A571, M1-G570, M1-G569, M1-M568, M1-L567, M1-Y566, M1-E565, M1-I564,M1-V563, M1-L562, M1-G561, M1-I560, M1-D559, M1-I558, M1-L557, M1-S556,M1-I555, M1-R554, M1-Y553, M1-D552, M1-P551, M1-P550, M1-L549, M1-N548,M1-G547, M1-K546, M1-K545, M1-V544, M1-D543, M1-R542, M1-V541, M1-L540,M1-H539, M1-Y538, M1-L537, M1-T536, M1-N535, M1-S534, M1-P533, M1-G532,M1-H531, M1-R530, M1-T529, M1-N528, M1-Y527, M1-L526, M1-E525, M1-E524,M1-L523, M1-R522, M1-S521, M1-I520, M1-T519, M1-L518, M1-F517, M1-R516,M1-H515, M1-M514, M1-S513, M1-V512, M1-G511, M1-N510, M1-E509, M1-I508,M1-L507, M1-L506, M1-K505, M1-V504, M1-F503, M1-D502, M1-V501, M1-R500,M1-D499, M1-L498, M1-V497, M1-L496, M1-A495, M1-D494, M1-L493, M1-M492,M1-A491, M1-Q490, M1-E489, M1-L488, M1-S487, M1-G486, M1-V485, M1-P484,M1-W483, M1-Q482, M1-Q481, M1-G480, M1-Y479, M1-I478, M1-F477, M1-I476,M1-Q475, M1-S474, M1-R473, M1-A472, M1-I471, M1-D470, M1-V469, M1-R468,M1-N467, M1-W466, M1-A465, M1-L464, M1-A463, M1-L462, M1-S461, M1-L460,M1-Q459, M1-D458, M1-P457, M1-A456, M1-S455, M1-A454, M1-N453, M1-A452,M1-G451, M1-K450, M1-L449, M1-L448, M1-A447, M1-T446, M1-L445, M1-I444,M1-A443, M1-L442, M1-D441, M1-I440, M1-D439, M1-Q438, M1-H437, M1-G436,M1-E435, M1-S434, M1-G433, M1-M432, M1-R431, M1-F430, M1-V429, M1-T428,M1-I427, M1-L426, M1-E425, M1-K424, M1-K423, M1-K422, M1-M421, M1-C420,M1-E419, M1-M418, M1-L417, M1-I416, M1-I415, M1-F414, M1-L413, M1-H412,M1-Q411, M1-A410, M1-Q409, M1-T408, M1-R407, M1-T406, M1-Y405, M1-T404,M1-F403, M1-T402, M1-K401, M1-Q400, M1-I399, M1-T398, M1-V397, M1-L396,M1-L395, M1-Q394, M1-D393, M1-R392, M1-L391, M1-S390, M1-E389, M1-N388,M1-I387, M1-L386, M1-G385, M1-G384, M1-E383, M1-E382, M1-S381, M1-Y380,M1-K379, M1-H378, M1-G377, M1-F376, M1-A375, M1-L374, M1-I373, M1-D372,M1-S371, M1-A370, M1-R369, M1-G368, M1-S367, M1-G366, M1-D365, M1-C364,M1-V363, M1-V362, M1-V361, M1-P360, M1-V359, M1-P358, M1-P357, M1-T356,M1-D355, M1-R354, M1-L353, M1-Y352, M1-E351, M1-L350, M1-V349, M1-I348,M1-S347, M1-I346, M1-V345, M1-N344, M1-P343, M1-G342, M1-G341, M1-E340,M1-V339, M1-I338, M1-L337, M1-A336, M1-V335, M1-V334, M1-P333, M1-V332,M1-G331, M1-Q330, M1-G329, M1-I328, M1-R327, M1-T326, M1-N325, M1-I324,M1-K323, M1-Q322, M1-L321, M1-S320, M1-I319, M1-H318, M1-K317, M1-E316,M1-L315, M1-Q314, M1-R313, M1-R312, M1-L311, M1-K310, M1-V309, M1-E308,M1-A307, M1-G306, M1-Y305, M1-K304, M1-G303, M1-T302, M1-T301, M1-G300,M1-N299, M1-D298, M1-A297, M1-L296, M1-I295, M1-F294, M1-H293, M1-S292,M1-H291, M1-M290, M1-S289, M1-N288, M1-L287, M1-V286, M1-T285, M1-L284,M1-K283, M1-S282, M1-M281, M1-P280, M1-N279, M1-S278, M1-M277, M1-T276,M1-Q275, M1-Y274, M1-P273, M1-R272, M1-V271, M1-V270, M1-D269, M1-R268,M1-G267, M1-I266, M1-L265, M1-D264, M1-E263, M1-Q262, M1-N261, M1-E260,M1-V259, M1-I258, M1-G257, M1-W256, M1-P255, M1-A254, M1-I253, M1-G252,M1-I251, M1-T250, M1-C249, M1-I248, M1-K247, M1-G246, M1-R245, M1-S244,M1-K243, M1-S242, M1-A241, M1-H240, M1-D239, M1-K238, M1-L237, M1-A236,M1-D235, M1-G234, M1-V233, M1-H232, M1-R231, M1-I230, M1-V229, M1-G228,M1-T227, M1-N226, M1-V225, M1-G224, M1-G223, M1-T222, M1-F221, M1-I220,M1-W219, M1-A218, M1-G217, M1-T216, M1-T215, M1-M214, M1-A213, M1-A212,M1-K211, M1-I210, M1-L209, M1-G208, M1-K207, M1-G206, M1-F205, M1-V204,M1-Q203, M1-K202, M1-L201, M1-K200, M1-P199, M1-Q198, M1-L197, M1-E196,M1-F195, M1-N194, M1-Q193, M1-L192, M1-G191, M1-G190, M1-H189, M1-V188,M1-S187, M1-I186, M1-L185, M1-L184, M1-K183, M1-P182, M1-L181, M1-E180,M1-L179, M1-Q178, M1-W177, M1-E176, M1-K175, M1-T174, M1-M173, M1-L172,M1-H171, M1-L170, M1-L169, M1-L168, M1-D167, M1-P166, M1-K165, M1-T164,M1-D163, M1-F162, M1-S161, M1-V160, M1-R159, M1-V158, M1-Y157, M1-M156,M1-A155, M1-K154, M1-N153, M1-S152, M1-H151, M1-G150, M1-G149, M1-G148,M1-Q147, M1-F146, M1-E145, M1-I144, M1-T143, M1-G142, M1-F141, M1-A140,M1-D139, M1-T138, M1-P137, M1-S136, M1-L135, M1-Q134, M1-T133, M1-H132,M1-K131, M1-S130, M1-I129, M1-S128, M1-W127, M1-K126, M1-E125, M1-S124,M1-Q123, M1-I122, M1-D121, M1-N120, M1-R119, M1-S118, M1-L117, M1-R116,M1-S115, M1-E114, M1-N113, M1-K112, M1-E111, M1-N110, M1-Q109, M1-L108,M1-V107, M1-S106, M1-I105, M1-S104, M1-P103, M1-T102, M1-L101, M1-G100,M1-V99, M1-H98, M1-Q97, M1-G96, M1-I95, M1-L94, M1-R93, M1-G92, M1-C91,M1-C90, M1-C89, M1-R88, M1-H87, M1-P86, M1-D85, M1-K84, M1-T83, M1-S82,M1-P81, M1-I80, M1-I79, M1-H78, M1-V77, M1-C76, M1-E75, M1-R74, M1-K73,M1-Y72, M1-F71, M1-A70, M1-R69, M1-E68, M1-I67, M1-W66, M1-S65, M1-K64,M1-Q63, M1-A62, M1-P61, M1-T60, M1-Q59, M1-Q58, M1-S57, M1-P56, M1-L55,M1-G54, M1-S53, M1-R52, M1-K51, M1-T50, M1-S49, M1-G48, M1-Q47, M1-K46,M1-S45, M1-S44, M1-L43, M1-R42, M1-K41, M1-W40, M1-S39, M1-E38, M1-A37,M1-F36, M1-R35, M1-G34, M1-R33, M1-S32, M1-A31, M1-S30, M1-R29, M1-S28,M1-R27, M1-R26, M1-R25, M1-S24, M1-E23, M1-A22, M1-N21, M1-D20, M1-E19,M1-R18, M1-D17, M1-S16, M1-C15, M1-G14, M1-R13, M1-E12, M1-M11, M1-E10,M1-A9, M1-A8, and/or M1-D7 of SEQ ID NO:2. Polynucleotide sequencesencoding these polypeptides are also provided. The present inventionalso encompasses the use of these C-terminal LTRPC3g deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

[0239] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3g polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3g polypeptide deletions) of SEQ ID NO:2.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3g (SEQ ID NO:2), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3g (SEQ ID NO:2).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0240] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5187 ofSEQ ID NO:1, b is an integer between 15 to 5201, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14.

[0241] Features of the Polypeptide Encoded by Gene No:2

[0242] The polypeptide of this gene provided as SEQ ID NO:4 (FIGS.2A-F), encoded by the polynucleotide sequence according to SEQ ID NO:3(FIGS. 2A-F), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3h, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3hpolypeptide with this protein is provided in FIGS. 7A-G.

[0243] The LTRPC3h polypeptide was determined to share 65.7% identityand 73.5% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0244] The LTRPC3h protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0245] Specifically, the LTRPC3h (SEQ ID NO:4) polypeptide represents anovel variant of the LTRPC3b (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:17; and also co-pending U.S. Ser. No. 10/405,793,filed Mar. 28, 2003; which are hereby incorporated by reference hereinin their entirety) polypeptide. The LTRPC3h represents a novel member ofthe TRPM subfamily.

[0246] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0247] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art, particularly LTRPC3b, and LTRPC3.

[0248] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3h polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 2A-F. The transmembrane domains arelocated from about amino acid 787 to about amino acid 804 (TM1), fromabout amino acid 884 to about amino acid 901 (TM2), from about aminoacid 957 to about amino acid 970 (TM3), from about amino acid 984 toabout amino acid 1001 (TM4), from about amino acid 1018 to about aminoacid 1035 (TM5), and/or from about amino acid 1106 to about amino acid1125 (TM6) of SEQ ID NO:4. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0249] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:36), IVKFWFYTLAYIGYLMLF (SEQ ID NO:37),VTDLIAILLFSVGM (SEQ ID NO:38), RVIYCVNIIYWYIRLLDI (SEQ ID NO:39),MMIDMMYFVIIMLVVLMS (SEQ ID NO:40), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:41). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3htransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0250] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3h, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3h polypeptide,particularly for the development of antibodies specific to theseregions.

[0251] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:42),NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:43),ILRLQDQPFRSDG (SEQ ID NO:44), FGVNKYLGPYVMMIGK (SEQ ID NO:45), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP(SEQ ID NO:46). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the LTRPC3hinter-transmembrane polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0252] The LTRPC3h polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 364, 420, 586, 749, 766, 988,1079, 1096, 1109, 1212, 1303, and 1564 of SEQ ID No: 4 (FIGS. 2A-F).Conservation of cysteines at key amino acid residues is indicative ofconserved structural features, which may correlate with conservation ofprotein function and/or activity.

[0253] In confirmation of the LTRPC3h representing a member of thetransient receptor channel family, the LTRPC3h polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1040 to about amino acid 1045 of SEQ ID NO:4. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0254] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:47).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3h TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0255] In further confirmation of the LTRPC3h representing a member ofthe transient receptor channel family, the LTRPC3h polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 915 to about amino acid 1126 of SEQ ID NO:4.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0256] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:48).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3h ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0257] The LTRPC3h polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1230 to aboutamino acid 1285 of SEQ ID NO:4. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0258] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:49).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3h coiled-coil domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0259] LTRPC3h polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3h by identifying mutations in the LTRPC3h gene using LTRPC3hsequences as probes or by determining LTRPC3h protein or mRNA expressionlevels. LTRPC3h polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3h peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3h.

[0260] Since the LTRPC3h polypeptide represents an N-terminally extendedvariant of the LTRPC3b polypeptide (see polypeptide alignment providedin FIG. 11A-I), and since the LTRPC3b (SEQ ID NO:17) polypeptiderepesents a splice variant of the LTRPC3 polypeptide (SEQ ID NO:15), theLTRPC3h (SEQ ID NO:4) polypeptide of the present invention is expectedto have the same or similar function as the LTRPC3 polypeptide, and inparticular, the LTRPC3b polypeptide. Specifically, the LTRPC3hpolypeptide is expected to localize to the cell membrane.

[0261] Moreover, the LTRPC3h polypeptide is expected to also localize inor near the plasmalemmal compartment.

[0262] Moreover, the LTRPC3h polypeptide is also expected to be aconstitutively active channel capable of mediating Ca²⁺ influx.

[0263] Moreover, the LTRPC3h polypeptide is also expected to mediateCa²⁺ entry and that LTRPC3h-mediated Ca²⁺ entry is expected to bepotentiated by store-depletion.

[0264] Moreover, the LTRPC3h polypeptide is also expected to mediate aCa²⁺ entry pathway that is distinct from the endogenous Ca²⁺ entrypathways present in HEK 293 cells.

[0265] Moreover, the LTRPC3h polypeptide of the present invention isexpected to share the same or similar expression profile as that of theLTRPC3 polypeptide (SEQ ID NO:15).

[0266] Moreover, the LTRPC3h polypeptide is expected to share the sameor similar differential expression patterns observed for the LTRPC3variant.

[0267] Moreover, the LTRCP3g polypeptide is expected to be able tomodulate the FEN1 DNA base-excision repair/proliferation modulatingprotein to the same or similar extent as the LTRPC3 polypeptide.

[0268] Moreover, the LTRPC3h polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein, such as LTRPC3 (SEQ ID NO:15), andparticularly as LTRPC3b (SEQ ID NO:17).

[0269] Moreover, the LTRPC3h polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein, such as the LTRPC3 variant (SEQ IDNO:15), and particularly as LTRPC3b (SEQ ID NO:17).

[0270] In preferred embodiments, LTRPC3h polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0271] In preferred embodiments, LTRPC3h polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0272] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0273] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0274] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1 modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3h to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3h activity or a compound that increased LTRPC3h message levelswould be a desired invention for cancer therapy.

[0275] In preferred embodiments, LTRPC3h polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0276] Moreover, LTRPC3h polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0277] In preferred embodiments, antagonists directed against LTRPC3hare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0278] Moreover, antagonists directed against LTRPC3h are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0279] In preferred embodiments, agonists directed against LTRPC3h areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0280] Moreover, agonists directed against LTRPC3h are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0281] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3hpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0282] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3): 127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0283] Thus, the LTRPC3h polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0284] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3hpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0285] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3hpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0286] In preferred embodiments, LTRPC3h polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3hpolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0287] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3h polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0288] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0289] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0290] Thus, the LTRPC3h polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0291] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0292] In preferred embodiments, LTRPC3h polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0293] In more preferred embodiments, LTRPC3h polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0294] LTRPC3h polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3hpolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0295] The LTRPC3h gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3h can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0296] Moreover, given the selective expression in kidney, LTRPC3h couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3h chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3h is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3h inthe disease. Therefore, it is possible that LTRPC3h may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0297] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3h may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3h may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0298] In addition, LTRPC3h polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0299] The LTRPC3h polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3h polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0300] In addition, antagonists of the LTRPC3h polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0301] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3h polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3h, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0302] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3h gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0303] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3h, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3h and assessing their ability to grow wouldprovide convincing evidence the LTRPC3h polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0304] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0305] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0306] In the case of LTRPC3h transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0307] In preferred embodiments, the following N-terminal LTRPC3hdeletion polypeptides are encompassed by the present invention:M1-T1721, G2-T1721, K3-T1721, K4-T1721, W5-T1721, R6-T1721, D7-T1721,A8-T1721, A9-T1721, E10-T1721, M11-T1721, E12-T1721, R13-T1721,G14-T1721, C15-T1721, S16-T1721, D17-T1721, R18-T1721, E19-T1721,D20-T1721, N21-T1721, A22-T1721, E23-T1721, S24-T1721, R25-T1721,R26-T1721, R27-T1721, S28-T1721, R29-T1721, S30-T1721, A31-T1721,S32-T1721, R33-T1721, G34-T1721, R35-T1721, F36-T1721, A37-T1721,E38-T1721, S39-T1721, W40-T1721, K41-T1721, R42-T1721, L43-T1721,S44-T1721, S45-T1721, K46-T1721, Q47-T1721, G48-T1721, S49-T1721,T50-T1721, K51-T1721, R52-T1721, S53-T1721, G54-T1721, L55-T1721,P56-T1721, S57-T1721, Q58-T1721, Q59-T1721, T60-T1721, P61-T1721,A62-T1721, Q63-T1721, K64-T1721, S65-T1721, W66-T1721, I67-T1721,E68-T1721, R69-T1721, A70-T1721, F71-T1721, Y72-T1721, K73-T1721,R74-T1721, E75-T1721, C76-T1721, V77-T1721, H78-T1721, I79-T1721,I80-T1721, P81-T1721, S82-T1721, T83-T1721, K84-T1721, D85-T1721,P86-T1721, H87-T1721, R88-T1721, C89-T1721, C90-T1721, C91-T1721,G92-T1721, R93-T1721, L94-T1721, I95-T1721, G96-T1721, Q97-T1721,H98-T1721, V99-T1721, G100-T1721, L10-T1721, T102-T1721, P103-T1721,S104-T1721, I105-T1721, S106-T1721, V107-T1721, L108-T1721, Q109-T1721,N110-T1721, E111-T1721, K112-T1721, N113-T1721, E114-T1721, S115-T1721,R116-T1721, L117-T1721, S118-T1721, R119-T1721, N120-T1721, D121-T1721,I122-T1721, Q123-T1721, S124-T1721, E125-T1721, K126-T1721, W127-T1721,S128-T1721, I129-T1721, S130-T1721, K131-T1721, H132-T1721, T133-T1721,Q134-T1721, L135-T1721, S136-T1721, P137-T1721, T138-T1721, D139-T1721,A140-T1721, F141-T1721, G142-T1721, T143-T1721, I144-T1721, E145-T1721,F146-T1721, Q147-T1721, G148-T1721, G149-T1721, G150-T1721, H151-T1721,S152-T1721, N153-T1721, K154-T1721, and/or A155-T1721 of SEQ ID NO:4.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3h deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0308] In preferred embodiments, the following C-terminal LTRPC3hdeletion polypeptides are encompassed by the present invention:M1-T1721, M1-H1720, M1-K1719, M1-S1718, M1-E1717, M1-F1716, M1-S1715,M1-Q1714, M1-F1713, M1-A1712, M1-S1711, M1-T1710, M1-R1709, M1-S1708,M1-L1707, M1-R1706, M1-R1705, M1-M1704, M1-S1703, M1-L1702, M1-S1701,M1-D1700, M1-G1699, M1-R1698, M1-G1697, M1-E1696, M1-P1695, M1-K1694,M1-S1693, M1-S1692, M1-K1691, M1-S1690, M1-R1689, M1-Q1688, M1-F1687,M1-P1686, M1-N1685, M1-Q1684, M1-L1683, M1-S1682, M1-A1681, M1-T1680,M1-N1679, M1-R1678, M1-Q1677, M1-R1676, M1-D1675, M1-L1674, M1-K1673,M1-D1672, M1-S1671, M1-I1670, M1-S1669, M1-F1668, M1-S1667, M1-K1666,M1-R1665, M1-T1664, M1-H1663, M1-A1662, M1-Y1661, M1-P1660, M1-A1659,M1-S1658, M1-P1657, M1-E1656, M1-E1655, M1-A1654, M1-S1653, M1-Y1652,M1-S1651, M1-N1650, M1-A1649, M1-R1648, M1-E1647, M1-I1646, M1-K1645,M1-P1644, M1-V1643, M1-T1642, M1-I1641, M1-N1640, M1-N1639, M1-S1638,M1-L1637, M1-T1636, M1-R1635, M1-E1634, M1-S1633, M1-N1632, M1-D1631,M1-G1630, M1-E1629, M1-Q1628, M1-S1627, M1-S1626, M1-I1625, M1-A1624,M1-I1623, M1-T1622, M1-A1621, M1-R1620, M1-R1619, M1-G1618, M1-K1617,M1-A1616, M1-E1615, M1-N1614, M1-E1613, M1-E1612, M1-S1611, M1-D1610,M1-S1609, M1-S1608, M1-P1607, M1-H1606, M1-S1605, M1-L1604, M1-E1603,M1-A1602, M1-E1601, M1-R1600, M1-E1599, M1-P1598, M1-H1597, M1-C1596,M1-C1595, M1-T1594, M1-L1593, M1-D1592, M1-E1591, M1-V1590, M1-K1589,M1-D1588, M1-G1587, M1-L1586, M1-G1585, M1-G1584, M1-P1583, M1-F1582,M1-A1581, M1-A1580, M1-R1579, M1-D1578, M1-A1577, M1-I1576, M1-A1575,M1-Q1574, M1-P1573, M1-A1572, M1-N1571, M1-V1570, M1-C1569, M1-R1568,M1-T1567, M1-D1566, M1-I1565, M1-C1564, M1-D1563, M1-T1562, M1-I1561,M1-S1560, M1-T1559, M1-Y1558, M1-E1557, M1-A1556, M1-T1555, M1-K1554,M1-V1553, M1-P1552, M1-V1551, M1-G1550, M1-F1549, M1-N1548, M1-A1547,M1-Y1546, M1-Y1545, M1-S1544, M1-R1543, M1-S1542, M1-P1541, M1-S1540,M1-F1539, M1-M1538, M1-F1537, M1-S1536, M1-H1535, M1-S1534, M1-K1533,M1-V1532, M1-I1531, M1-P1530, M1-A1529, M1-E1528, M1-E1527, M1-L1526,M1-L1525, M1-F1524, M1-P1523, M1-T1522, M1-T1521, M1-A1520, M1-L1519,M1-Y1518, M1-R1517, M1-S1516, M1-S1515, M1-K1514, M1-S1513, M1-R1512,M1-E1511, M1-I1510, M1-T1509, M1-H1508, M1-Y1507, M1-M1506, M1-P1505,M1-P1504, M1-E1503, M1-S1502, M1-D1501, M1-W1500, M1-P1499, M1-N1498,M1-Q1497, M1-C1496, M1-E1495, M1-P1494, M1-L1493, M1-H1492, M1-T1491,M1-Y1490, M1-D1489, M1-S1488, M1-S1487, M1-F1486, M1-S1485, M1-R1484,M1-T1483, M1-D1482, M1-M1481, M1-S1480, M1-T1479, M1-I1478, M1-D1477,M1-E1476, M1-F1475, M1-D1474, M1-I1473, M1-S1472, M1-R1471, M1-S1470,M1-P1469, M1-P1468, M1-R1467, M1-D1466, M1-T1465, M1-P1464, M1-A1463,M1-L1462, M1-T1461, M1-A1460, M1-Y1459, M1-A1458, M1-S1457, M1-S1456,M1-S1455, M1-P1454, M1-A1453, M1-T1452, M1-S1451, M1-P1450, M1-V1449,M1-P1448, M1-T1447, M1-S1446, M1-F1445, M1-S1444, M1-P1443, M1-E1442,M1-G1441, M1-L1440, M1-G1439, M1-L1438, M1-I1437, M1-N1436, M1-V1435,M1-S1434, M1-N1433, M1-D1432, M1-L1431, M1-P1430, M1-D1429, M1-I1428,M1-D1427, M1-C1426, M1-H1425, M1-L1424, M1-E1423, M1-D1422, M1-M1421,M1-A1420, M1-S1419, M1-V1418, M1-Y1417, M1-I1416, M1-D1415, M1-I1414,M1-C1413, M1-S1412, M1-S1411, M1-P1410, M1-R1409, M1-R1408, M1-S1407,M1-D1406, M1-P1405, M1-V1404, M1-I1403, M1-A1402, M1-L1401, M1-T1400,M1-N1399, M1-A1398, M1-P1397, M1-A1396, M1-A1395, M1-P1394, M1-A1393,M1-K1392, M1-P1391, M1-E1390, M1-K1389, M1-A1388, M1-V1387, M1-S1386,M1-H1385, M1-S1384, M1-S1383, M1-T1382, M1-A1381, M1-R1380, M1-H1379,M1-L1378, M1-S1377, M1-L1376, M1-S1375, M1-R1374, M1-E1373, M1-K1372,M1-F1371, M1-I1370, M1-S1369, M1-E1368, M1-L1367, M1-K1366, M1-E1365,M1-I1364, M1-G1363, M1-G1362, M1-K1361, M1-D1360, M1-K1359, M1-M1358,M1-N1357, M1-V1356, M1-S1355, M1-Y1354, M1-F1353, M1-S1352, M1-H1351,M1-S1350, M1-R1349, M1-M1348, M1-R1347, M1-P1346, M1-M1345, M1-L1344,M1-T1343, M1-P1342, M1-S1341, M1-T1340, M1-P1339, M1-S1338, M1-M1337,M1-T1336, M1-E1335, M1-E1334, M1-G1333, M1-A1332, M1-P1331, M1-D1330,M1-I1329, M1-S1328, M1-E1327, M1-Q1326, M1-L1325, M1-K1324, M1-F1323,M1-T1322, M1-N1321, M1-G1320, M1-E1319, M1-Q1318, M1-S1317, M1-N1316,M1-F1315, M1-S1314, M1-S1313, M1-Q1312, M1-R1311, M1-V1310, M1-I1309,M1-Y1308, M1-A1307, M1-A1306, M1-D1305, M1-T1304, M1-C1303, M1-D1302,M1-S1301, M1-S1300, M1-T1299, M1-R1298, M1-S1297, M1-R1296, M1-I1295,M1-K1294, M1-N1293, M1-S1292, M1-E1291, M1-A1290, M1-R1289, M1-E1288,M1-L1287, M1-G1286, M1-T1285, M1-L1284, M1-R1283, M1-E1282, M1-L1281,M1-A1280, M1-T1279, M1-A1278, M1-M1277, M1-R1276, M1-G1275, M1-I1274,M1-L1273, M1-D1272, M1-E1271, M1-L1270, M1-Q1269, M1-A1268, M1-L1267,M1-R1266, M1-I1265, M1-D1264, M1-V1263, M1-T1262, M1-Q1261, M1-L1260,M1-S1259, M1-A1258, M1-K1257, M1-M1256, M1-S1255, M1-H1254, M1-E1253,M1-R1252, M1-E1251, M1-N1250, M1-V1249, M1-E1248, M1-E1247, M1-L1246,M1-R1245, M1-M1244, M1-S1243, M1-M1242, M1-N1241, M1-E1240, M1-V1239,M1-R1238, M1-E1237, M1-S1236, M1-T1235, M1-V1234, M1-R1233, M1-I1232,M1-R1231, M1-E1230, M1-D1229, M1-N1228, M1-S1227, M1-S1226, M1-N1225,M1-F1224, M1-R1223, M1-D1222, M1-D1221, M1-K1220, M1-E1219, M1-R1218,M1-F1217, M1-Y1216, M1-E1215, M1-E1214, M1-I1213, M1-C1212, M1-Q1211,M1-E1210, M1-E1209, M1-F1208, M1-D1207, M1-H1206, M1-V1205, M1-K1204,M1-K1203, M1-L1202, M1-E1201, M1-D1200, M1-D1199, M1-T1198, M1-I1197,M1-F1196, M1-L1195, M1-K1194, M1-L1193, M1-G1192, M1-Y1191, M1-D1190,M1-R1189, M1-E1188, M1-D1187, M1-P1186, M1-D1185, M1-S1184, M1-E1183,M1-H1182, M1-K1181, M1-R1180, M1-W1179, M1-R1178, M1-C1177, M1-C1176,M1-L1175, M1-H1174, M1-Q1173, M1-F1172, M1-I1171, M1-M1170, M1-T1169,M1-M1168, M1-H1167, M1-S1166, M1-F1165, M1-I1164, M1-I1163, M1-L1162,M1-P1161, M1-P1160, M1-P1159, M1-L1158, M1-V1157, M1-P1156, M1-R1155,M1-E1154, M1-H1153, M1-F1152, M1-T1151, M1-M1150, M1-I1149, M1-L1148,M1-Q1147, M1-Y1146, M1-R1145, M1-Q1144, M1-F1143, M1-K1142, M1-W1141,M1-V1140, M1-Q1139, M1-N1138, M1-S1137, M1-I1136, M1-S1135, M1-K1134,M1-V1133, M1-E1132, M1-F1131, M1-F1130, M1-T1129, M1-N1128, M1-N1127,M1-F1126, M1-V1125, M1-A1124, M1-I1123, M1-L1122, M1-L1121, M1-N1120,M1-V1119, M1-L1118, M1-L1117, M1-I1116, M1-N1115, M1-A1114, M1-V1113,M1-L1112, M1-L1111, M1-Y1110, M1-C1109, M1-A1108, M1-M1107, M1-I1106,M1-A1105, M1-P1104, M1-V1103, M1-I1102, M1-W1110, M1-A1100, M1-G1099,M1-T1098, M1-K1097, M1-C1096, M1-P1095, M1-P1094, M1-L1093, M1-Q1092,M1-I1091, M1-I1090, M1-K1089, M1-G1088, M1-D1087, M1-E1086, M1-R1085,M1-T1084, M1-E1083, M1-N1082, M1-Q1081, M1-G1080, M1-C1079, M1-P1078,M1-P1077, M1-D1076, M1-I1075, M1-Q1074, M1-D1073, M1-A1072, M1-F1071,M1-V1070, M1-E1069, M1-G1068, M1-Y1067, M1-I1066, M1-M1065, M1-W1064,M1-Y1063, M1-P1062, M1-M1061, M1-Y1060, M1-F1059, M1-I1058, M1-N1057,M1-K1056, M1-A1055, M1-L1054, M1-K1053, M1-W1052, M1-S1051, M1-P1050,M1-E1049, M1-E1048, M1-N1047, M1-P1046, M1-F1045, M1-L1044, M1-I1043,M1-A1042, M1-Q1041, M1-R1040, M1-A1039, M1-V1038, M1-G1037, M1-F1036,M1-S1035, M1-M1034, M1-L1033, M1-V1032, M1-V1031, M1-L1030, M1-M1029,M1-I1028, M1-I1027, M1-V1026, M1-F1025, M1-Y1024, M1-M1023, M1-M1022,M1-D1021, M1-I1020, M1-M1019, M1-M1018, M1-K1017, M1-G1016, M1-I1015,M1-M1014, M1-M1013, M1-V1012, M1-Y1011, M1-P101, M1-G1009, M1-L1008,M1-Y1007, M1-K1006, M1-N1005, M1-V1004, M1-G1003, M1-F1002, M1-I1001,M1-D1000, M1-L999, M1-L998, M1-R997, M1-I996, M1-Y995, M1-W994, M1-Y993,M1-I992, M1-I991, M1-N990, M1-V989, M1-C988, M1-Y987, M1-I986, M1-V985,M1-R984, M1-G983, M1-D982, M1-S981, M1-R980, M1-F979, M1-P978, M1-Q977,M1-D976, M1-Q975, M1-L974, M1-R973, M1-L972, M1-I971, M1-M970, M1-G969,M1-V968, M1-S967, M1-F966, M1-L965, M1-L964, M1-I963, M1-A962, M1-I961,M1-L960, M1-D959, M1-T958, M1-V957, M1-N956, M1-W955, M1-Y954, M1-E953,M1-Q952, M1-L951, M1-W950, M1-V949, M1-K948, M1-V947, M1-K946, M1-Q945,M1-L944, M1-L943, M1-K942, M1-G941, M1-P940, M1-E939, M1-S938, M1-M937,M1-L936, M1-I935, M1-E934, M1-R933, M1-M932, M1-K931, M1-E930, M1-I929,M1-G928, M1-L927, M1-T926, M1-F925, M1-I924, M1-Y923, M1-S922, M1-I921,M1-V920, M1-I919, M1-W918, M1-E917, M1-Q916, M1-T915, M1-S914, M1-P913,M1-W912, M1-R911, M1-E910, M1-M909, M1-K908, M1-V907, M1-L906, M1-V905,M1-I904, M1-Y903, M1-N902, M1-F901, M1-L900, M1-M899, M1-L898, M1-Y897,M1-G896, M1-I895, M1-Y894, M1-A893, M1-L892, M1-T891, M1-Y890, M1-F889,M1-W888, M1-F887, M1-K886, M1-V885, M1-I884, M1-P883, M1-A882, M1-N881,M1-Y880, M1-F879, M1-E878, M1-Y877, M1-I876, M1-K875, M1-R874, M1-G873,M1-L872, M1-P871, M1-I870, M1-L869, M1-R868, M1-H867, M1-K866, M1-S865,M1-Q864, M1-V863, M1-E862, M1-E861, M1-E860, M1-D859, M1-K858, M1-K857,M1-R856, M1-S855, M1-S854, M1-E853, M1-G852, M1-N851, M1-N850, M1-R849,M1-G848, M1-L847, M1-M846, M1-A845, M1-T844, M1-L843, M1-E842, M1-M841,M1-D840, M1-E839, M1-E838, M1-E837, M1-K836, M1-E835, M1-K834, M1-T833,M1-P832, M1-K831, M1-E830, M1-P829, M1-E828, M1-E827, M1-A826, M1-E825,M1-K824, M1-E823, M1-Q822, M1-L821, M1-H820, M1-I819, M1-E818, M1-Q817,M1-A816, M1-Q815, M1-S814, M1-M813, M1-Y812, M1-P811, M1-M810, M1-D809,M1-D808, M1-K807, M1-N806, M1-K805, M1-F804, M1-E803, M1-L802, M1-S801,M1-L800, M1-I799, M1-S798, M1-P797, M1-P796, M1-L795, M1-L794, M1-I793,M1-G792, M1-L791, M1-I790, M1-V789, M1-K788, M1-L787, M1-G786, M1-S785,M1-N784, M1-K783, M1-R782, M1-M781, M1-R780, M1-L779, M1-R778, M1-G777,M1-M776, M1-W775, M1-M774, M1-D773, M1-T772, M1-L771, M1-L770, M1-M769,M1-Q768, M1-S767, M1-C766, M1-T765, M1-H764, M1-A763, M1-I762, M1-F761,M1-D760, M1-R759, M1-H758, M1-K757, M1-A756, M1-A755, M1-V754, M1-A753,M1-L752, M1-Q751, M1-L750, M1-C749, M1-T748, M1-A747, M1-N746, M1-S745,M1-W744, M1-N743, M1-K742, M1-L741, M1-E740, M1-Y739, M1-T738, M1-L737,M1-L736, M1-K735, M1-M734, M1-A733, M1-L732, M1-Q731, M1-E730, M1-D729,M1-Q728, M1-K727, M1-Y726, M1-S725, M1-Q724, M1-D723, M1-L722, M1-L721,M1-E720, M1-V719, M1-A718, M1-L717, M1-Q716, M1-G715, M1-F714, M1-D713,M1-R712, M1-S711, M1-N710, M1-H709, M1-N708, M1-L707, M1-E706, M1-Q705,M1-S704, M1-I703, M1-D702, M1-D701, M1-V700, M1-M699, M1-D698, M1-N697,M1-E696, M1-S695, M1-A694, M1-E693, M1-H692, M1-A691, M1-M690, M1-A689,M1-K688, M1-C687, M1-L686, M1-K685, M1-C684, M1-A683, M1-V682, M1-L681,M1-A680, M1-K679, M1-A678, M1-M677, M1-A676, M1-E675, M1-E674, M1-G673,M1-H672, M1-Q671, M1-W670, M1-F669, M1-F668, M1-L667, M1-A666, M1-M665,M1-K664, M1-Q663, M1-R662, M1-K661, M1-M660, M1-L659, M1-V658, M1-A657,M1-W656, M1-V655, M1-M654, M1-L653, M1-E652, M1-H651, M1-F650, M1-P649,M1-F648, M1-P647, M1-F646, M1-H645, M1-N644, M1-I643, M1-E642, M1-P641,M1-D640, M1-D639, M1-L638, M1-D637, M1-I636, M1-D635, M1-V634, M1-E633,M1-E632, M1-E631, M1-R630, M1-K629, M1-K628, M1-T627, M1-T626, M1-K625,M1-R624, M1-G623, M1-R622, M1-R621, M1-L620, M1-P619, M1-I618, M1-D617,M1-D616, M1-E615, M1-M614, M1-G613, M1-L612, M1-L611, M1-K610, M1-L609,M1-A608, M1-K607, M1-P606, M1-R605, M1-K604, M1-P603, M1-G602, M1-F601,M1-L600, M1-N599, M1-H598, M1-Y597, M1-L596, M1-T595, M1-R594, M1-F593,M1-R592, M1-K591, M1-R590, M1-T589, M1-Y588, M1-N587, M1-C586, M1-R585,M1-Y584, M1-A583, M1-G582, M1-G581, M1-M580, M1-L579, M1-Y578, M1-E577,M1-I576, M1-V575, M1-L574, M1-G573, M1-I572, M1-D571, M1-I570, M1-L569,M1-S568, M1-I567, M1-R566, M1-Y565, M1-D564, M1-P563, M1-P562, M1-L561,M1-N560, M1-G559, M1-K558, M1-F557, M1-Y556, M1-I555, M1-W554, M1-G553,M1-F552, M1-G551, M1-P550, M1-Y549, M1-E548, M1-R547, M1-K546, M1-K545,M1-V544, M1-D543, M1-R542, M1-V541, M1-L540, M1-H539, M1-Y538, M1-L537,M1-T536, M1-N535, M1-S534, M1-P533, M1-G532, M1-H531, M1-R530, M1-T529,M1-N528, M1-Y527, M1-L526, M1-E525, M1-E524, M1-L523, M1-R522, M1-S521,M1-I520, M1-T519, M1-L518, M1-F517, M1-R516, M1-H515, M1-M514, M1-S513,M1-V512, M1-G511, M1-N510, M1-E509, M1-I508, M1-L507, M1-L506, M1-K505,M1-V504, M1-F503, M1-D502, M1-V501, M1-R500, M1-D499, M1-L498, M1-V497,M1-L496, M1-A495, M1-D494, M1-L493, M1-M492, M1-A491, M1-Q490, M1-E489,M1-L488, M1-S487, M1-G486, M1-V485, M1-P484, M1-W483, M1-Q482, M1-Q481,M1-G480, M1-Y479, M1-I478, M1-F477, M1-I476, M1-Q475, M1-S474, M1-R473,M1-A472, M1-I471, M1-D470, M1-V469, M1-R468, M1-N467, M1-W466, M1-A465,M1-L464, M1-A463, M1-L462, M1-S461, M1-L460, M1-Q459, M1-D458, M1-P457,M1-A456, M1-S455, M1-A454, M1-N453, M1-A452, M1-G451, M1-K450, M1-L449,M1-L448, M1-A447, M1-T446, M1-L445, M1-I444, M1-A443, M1-L442, M1-D441,M1-I440, M1-D439, M1-Q438, M1-H437, M1-G436, M1-E435, M1-S434, M1-G433,M1-M432, M1-R431, M1-F430, M1-V429, M1-T428, M1-I427, M1-L426, M1-E425,M1-K424, M1-K423, M1-K422, M1-M421, M1-C420, M1-E419, M1-M418, M1-L417,M1-I416, M1-I415, M1-F414, M1-L413, M1-H412, M1-Q411, M1-A410, M1-Q409,M1-T408, M1-R407, M1-T406, M1-Y405, M1-T404, M1-F403, M1-T402, M1-K401,M1-Q400, M1-I399, M1-T398, M1-V397, M1-L396, M1-L395, M1-Q394, M1-D393,M1-R392, M1-L391, M1-S390, M1-E389, M1-N388, M1-I387, M1-L386, M1-G385,M1-G384, M1-E383, M1-E382, M1-S381, M1-Y380, M1-K379, M1-H378, M1-G377,M1-F376, M1-A375, M1-L374, M1-I373, M1-D372, M1-S371, M1-A370, M1-R369,M1-G368, M1-S367, M1-G366, M1-D365, M1-C364, M1-V363, M1-V362, M1-V361,M1-P360, M1-V359, M1-P358, M1-P357, M1-T356, M1-D355, M1-R354, M1-L353,M1-Y352, M1-E351, M1-L350, M1-V349, M1-I348, M1-S347, M1-I346, M1-V345,M1-N344, M1-P343, M1-G342, M1-G341, M1-E340, M1-V339, M1-I338, M1-L337,M1-A336, M1-V335, M1-V334, M1-P333, M1-V332, M1-G331, M1-Q330, M1-G329,M1-I328, M1-R327, M1-T326, M1-N325, M1-I324, M1-K323, M1-Q322, M1-L321,M1-S320, M1-I319, M1-H318, M1-K317, M1-E316, M1-L315, M1-Q314, M1-R313,M1-R312, M1-L311, M1-K310, M1-V309, M1-E308, M1-A307, M1-G306, M1-Y305,M1-K304, M1-G303, M1-T302, M1-T301, M1-G300, M1-N299, M1-D298, M1-A297,M1-L296, M1-I295, M1-F294, M1-H293, M1-S292, M1-H291, M1-M290, M1-S289,M1-N288, M1-L287, M1-V286, M1-T285, M1-L284, M1-K283, M1-S282, M1-M281,M1-P280, M1-N279, M1-S278, M1-M277, M1-T276, M1-Q275, M1-Y274, M1-P273,M1-R272, M1-V271, M1-V270, M1-D269, M1-R268, M1-G267, M1-I266, M1-L265,M1-D264, M1-E263, M1-Q262, M1-N261, M1-E260, M1-V259, M1-I258, M1-G257,M1-W256, M1-P255, M1-A254, M1-I253, M1-G252, M1-I251, M1-T250, M1-C249,M1-I248, M1-K247, M1-G246, M1-R245, M1-S244, M1-K243, M1-S242, M1-A241,M1-H240, M1-D239, M1-K238, M1-L237, M1-A236, M1-D235, M1-G234, M1-V233,M1-H232, M1-R231, M1-I230, M1-V229, M1-G228, M1-T227, M1-N226, M1-V225,M1-G224, M1-G223, M1-T222, M1-F221, M1-I220, M1-W219, M1-A218, M1-G217,M1-T216, M1-T215, M1-M214, M1-A213, M1-A212, M1-K211, M1-I210, M1-L209,M1-G208, M1-K207, M1-G206, M1-F205, M1-V204, M1-Q203, M1-K202, M1-L201,M1-K200, M1-P199, M1-Q198, M1-L197, M1-E196, M1-F195, M1-N194, M1-Q193,M1-L192, M1-G191, M1-G190, M1-H189, M1-V188, M1-S187, M1-I186, M1-L185,M1-L184, M1-K183, M1-P182, M1-L181, M1-E180, M1-L179, M1-Q178, M1-W177,M1-E176, M1-K175, M1-T174, M1-M173, M1-L172, M1-H171, M1-L170, M1-L169,M1-L168, M1-D167, M1-P166, M1-K165, M1-T164, M1-D163, M1-F162, M1-S161,M1-V160, M1-R159, M1-V158, M1-Y157, M1-M156, M1-A155, M1-K154, M1-N153,M1-S152, M1-H151, M1-G150, M1-G149, M1-G148, M1-Q147, M1-F146, M1-E145,M1-I144, M1-T143, M1-G142, M1-F141, M1-A140, M1-D139, M1-T138, M1-P137,M1-S136, M1-L135, M1-Q134, M1-T133, M1-H132, M1-K131, M1-S130, M1-I129,M1-S128, M1-W127, M1-K126, M1-E125, M1-S124, M1-Q123, M1-I122, M1-D121,M1-N120, M1-R119, M1-S118, M1-L117, M1-R116, M1-S115, M1-E114, M1-N113,M1-K112, M1-E111, M1-N110, M1-Q109, M1-L108, M1-V107, M1-S106, M1-I05,M1-S104, M1-P103, M1-T102, M1-L101, M1-G100, M1-V99, M1-H98, M1-Q97,M1-G96, M1-I95, M1-L94, M1-R93, M1-G92, M1-C91, M1-C90, M1-C89, M1-R88,M1-H87, M1-P86, M1-D85, M1-K84, M1-T83, M1-S82, M1-P81, M1-I80, M1-I79,M1-H78, M1-V77, M1-C76, M1-E75, M1-R74, M1-K73, M1-Y72, M1-F71, M1-A70,M1-R69, M1-E68, M1-I67, M1-W66, M1-S65, M1-K64, M1-Q63, M1-A62, M1-P61,M1-T60, M1-Q59, M1-Q58, M1-S57, M1-P56, M1-L55, M1-G54, M1-S53, M1-R52,M1-K51, M1-T50, M1-S49, M1-G48, M1-Q47, M1-K46, M1-S45, M1-S44, M1-L43,M1-R42, M1-K41, M1-W40, M1-S39, M1-E38, M1-A37, M1-F36, M1-R35, M1-G34,M1-R33, M1-S32, M1-A31, M1-S30, M1-R29, M1-S28, M1-R27, M1-R26, M1-R25,M1-S24, M1-E23, M1-A22, M1-N21, M1-D20, M1-E19, M1-R18, M1-D17, M1-S16,M1-C15, M1-G14, M1-R13, M1-E12, M1-M11, M1-E10, M1-A9, M1-A8, and/orM1-D7 of SEQ ID NO:4. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal LTRPC3h deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0309] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3h polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3h polypeptide deletions) of SEQ ID NO:4.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3h (SEQ ID NO:4), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3h (SEQ ID NO:4).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0310] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:3 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5223 ofSEQ ID NO:3, b is an integer between 15 to 5237, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:3,and where b is greater than or equal to a+14.

[0311] Features of the Polypeptide Encoded by Gene No:3

[0312] The polypeptide of this gene provided as SEQ ID NO:6 (FIGS.3A-F), encoded by the polynucleotide sequence according to SEQ ID NO:5(FIGS. 3A-F), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3i, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3ipolypeptide with this protein is provided in FIGS. 7A-G.

[0313] The LTRPC3i polypeptide was determined to share 65.4% identityand 73.2% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0314] The LTRPC3i protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0315] Specifically, the LTRPC3i (SEQ ID NO:6) polypeptide represents anovel variant of the LTRPC3c (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:18; and also co-pending U.S. Ser. No. 10/405,793,filed Mar. 28, 2003; which are hereby incorporated by reference hereinin their entirety) polypeptide. The LTRPC3i represents a novel member ofthe TRPM subfamily.

[0316] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0317] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art, particularly LTRPC3c, and LTRPC3.

[0318] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3i polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 3A-F. The transmembrane domains arelocated from about amino acid 775 to about amino acid 792 (TM1), fromabout amino acid 872 to about amino acid 888 (TM2), from about aminoacid 945 to about amino acid 958 (TM3), from about amino acid 972 toabout amino acid 988 (TM4), from about amino acid 1006 to about aminoacid 1023 (TM5), and/or from about amino acid 1115 to about amino acid1125 (TM6) of SEQ ID NO:6. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0319] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:50), IVKFWFYTLAYIGYLMLF (SEQ ID NO:51),VTDLIAILLFSVGM (SEQ ID NO:52), RVIYCVNIIYWYIRLLDI (SEQ ID NO:53),MMIDMMYFVIIMLVVLMS (SEQ ID NO:54), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:55). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3itransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0320] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3i, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3i polypeptide,particularly for the development of antibodies specific to theseregions.

[0321] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:56),NYIVLVKMERWPSTQEWIVISYIFTLGEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:57),ILRLQDQPFRSDG (SEQ ID NO:58), FGVNKYLGPYVMMIGK (SEQ ID NO:59), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP(SEQ ID NO:60). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of the LTRPC3iinter-transmembrane polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0322] The LTRPC3i polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 364, 420, 574, 672, 754, 976,1212, 1303, and 1564 of SEQ ID NO:6 (FIGS. 3A-F). Conservation ofcysteines at key amino acid residues is indicative of conservedstructural features, which may correlate with conservation of proteinfunction and/or activity.

[0323] In confirmation of the LTRPC3i representing a member of thetransient receptor channel family, the LTRPC3i polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1140 to about amino acid 1145of SEQ ID NO:6. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0324] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:61).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3i TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0325] In further confirmation of the LTRPC3i representing a member ofthe transient receptor channel family, the LTRPC3i polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 915 to about amino acid 1126 of SEQ ID NO:6.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0326] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:62).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3i ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0327] The LTRPC3i polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1230 to aboutamino acid 1285 of SEQ ID NO:6. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0328] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:63).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3i coiled-coil domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0329] LTRPC3i polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3i by identifying mutations in the LTRPC3i gene using LTRPC3isequences as probes or by determining LTRPC3i protein or mRNA expressionlevels. LTRPC3i polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3i peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3i.

[0330] Since the LTRPC3i polypeptide represents an N-terminally extendedvariant of the LTRPC3c polypeptide (see polypeptide alignment providedin FIG. 11A-I), and since the LTRPC3c (SEQ ID NO:18) polypeptiderepesents a splice variant of the LTRPC3 polypeptide (SEQ ID NO:15), theLTRPC3i (SEQ ID NO:6) polypeptide of the present invention is expectedto have the same or similar function as the LTRPC3 polypeptide, and inparticular, the LTRPC3c polypeptide. Specifically, the LTRPC3ipolypeptide is expected to localize to the cell membrane.

[0331] Moreover, the LTRPC3i polypeptide is expected to also localize inor near the plasmalemmal compartment.

[0332] Moreover, the LTRPC3i polypeptide is also expected to be aconstitutively active channel capable of mediating Ca²⁺ influx.

[0333] Moreover, the LTRPC3i polypeptide is also expected to mediateCa²⁺ entry and that LTRPC3i-mediated Ca²⁺ entry is expected to bepotentiated by store-depletion.

[0334] Moreover, the LTRPC3i polypeptide is also expected to mediate aCa²⁺ entry pathway that is distinct from the endogenous Ca²⁺ entrypathways present in HEK 293 cells.

[0335] Moreover, the LTRPC3i polypeptide of the present invention isexpected to share the same or similar expression profile as that of theLTRPC3 polypeptide (SEQ ID NO:15).

[0336] Moreover, the LTRPC3i polypeptide is expected to share the sameor similar differential expression patterns observed for the LTRPC3variant.

[0337] Moreover, the LTRCP3g polypeptide is expected to be able tomodulate the FEN1 DNA base-excision repair/proliferation modulatingprotein to the same or similar extent as the LTRPC3 polypeptide.

[0338] Moreover, the LTRPC3i polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein, such as LTRPC3 (SEQ ID NO:15), andparticularly as LTRPC3c (SEQ ID NO:18).

[0339] Moreover, the LTRPC3i polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein, such as the LTRPC3 variant (SEQ IDNO:15), and particularly as LTRPC3c (SEQ ID NO:18).

[0340] In preferred embodiments, LTRPC3i polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0341] In preferred embodiments, LTRPC3i polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0342] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0343] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0344] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1 modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3i to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3i activity or a compound that increased LTRPC3i message levelswould be a desired invention for cancer therapy.

[0345] In preferred embodiments, LTRPC3i polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0346] Moreover, LTRPC3i polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0347] In preferred embodiments, antagonists directed against LTRPC3iare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0348] Moreover, antagonists directed against LTRPC3i are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0349] In preferred embodiments, agonists directed against LTRPC3i areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0350] Moreover, agonists directed against LTRPC3i are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0351] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3ipolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0352] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0353] Thus, the LTRPC3i polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0354] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3ipolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0355] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3ipolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0356] In preferred embodiments, LTRPC3i polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3ipolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0357] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3i polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0358] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0359] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0360] Thus, the LTRPC3i polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0361] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0362] In preferred embodiments, LTRPC3i polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0363] In more preferred embodiments, LTRPC3i polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0364] LTRPC3i polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3ipolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0365] The LTRPC3i gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3i can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0366] Moreover, given the selective expression in kidney, LTRPC3i couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3i chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3i is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3i inthe disease. Therefore, it is possible that LTRPC3i may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0367] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3i may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3i may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0368] In addition, LTRPC3i polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0369] The LTRPC3i polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3i polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0370] In addition, antagonists of the LTRPC3i polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0371] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3i polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3i, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0372] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3i gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0373] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3i, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3i and assessing their ability to grow wouldprovide convincing evidence the LTRPC3i polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0374] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0375] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0376] In the case of LTRPC3i transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0377] In preferred embodiments, the following N-terminal LTRPC3ideletion polypeptides are encompassed by the present invention:M1-T1721, G2-T1721, K3-T1721, K4-T1721, W5-T1721, R6-T1721, D7-T1721,A8-T1721, A9-T1721, E10-T1721, M11-T1721, E12-T1721, R13-T1721,G14-T1721, C15-T1721, S16-T1721, D17-T1721, R18-T1721, E19-T1721,D20-T1721, N21-T1721, A22-T1721, E23-T1721, S24-T1721, R25-T1721,R26-T1721, R27-T1721, S28-T1721, R29-T1721, S30-T1721, A31-T1721,S32-T1721, R33-T1721, G34-T1721, R35-T1721, F36-T1721, A37-T1721,E38-T1721, S39-T1721, W40-T1721, K41-T1721, R42-T1721, L43-T1721,S44-T1721, S45-T1721, K46-T1721, Q47-T1721, G48-T1721, S49-T1721,T50-T1721, K51-T1721, R52-T1721, S53-T1721, G54-T1721, L55-T1721,P56-T1721, S57-T1721, Q58-T1721, Q59-T1721, T60-T1721, P61-T1721,A62-T1721, Q63-T1721, K64-T1721, S65-T1721, W66-T1721, I67-T1721,E68-T1721, R69-T1721, A70-T1721, F71-T1721, Y72-T1721, K73-T1721,R74-T1721, E75-T1721, C76-T1721, V77-T1721, H78-T1721, I79-T1721,I80-T1721, P81-T1721, S82-T1721, T83-T1721, K84-T1721, D85-T1721,P86-T1721, H87-T1721, R88-T1721, C89-T1721, C90-T1721, C91-T1721,G92-T1721, R93-T1721, L94-T1721, I95-T1721, G96-T1721, Q97-T1721,H98-T1721, V99-T1721, G100-T1721, L10-T1721, T102-T1721, P103-T1721,S104-T1721, I105-T1721, S106-T1721, V107-T1721, L108-T1721, Q109-T1721,N110-T1721, E111-T1721, K112-T1721, N113-T1721, E114-T1721, S115-T1721,R116-T1721, L117-T1721, S118-T1721, R119-T1721, N120-T1721, D121-T1721,I122-T1721, Q123-T1721, S124-T1721, E125-T1721, K126-T1721, W127-T1721,S128-T1721, I129-T1721, S130-T1721, K131-T1721, H132-T1721, T133-T1721,Q134-T1721, L135-T1721, S136-T1721, P137-T1721, T138-T1721, D139-T1721,A140-T1721, F141-T1721, G142-T1721, T143-T1721, I144-T1721, E145-T1721,F146-T1721, Q147-T1721, G148-T1721, G149-T1721, G150-T1721, H151-T1721,S152-T1721, N153-T1721, K154-T1721, and/or A155-T1721 of SEQ ID NO:6.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3i deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0378] In preferred embodiments, the following C-terminal LTRPC3ideletion polypeptides are encompassed by the present invention:M1-T1721, M1-H1720, M1-K1719, M1-S1718, M1-E1717, M1-F1716, M1-S1715,M1-Q1714, M1-F1713, M1-A1712, M1-S1711, M1-T1710, M1-R1709, M1-S1708,M1-L1707, M1-R1706, M1-R1705, M1-M1704, M1-S1703, M1-L1702, M1-S1701,M1-D1700, M1-G1699, M1-R1698, M1-G1697, M1-E1696, M1-P1695, M1-K1694,M1-S1693, M1-S1692, M1-K1691, M1-S1690, M1-R1689, M1-Q1688, M1-F1687,M1-P1686, M1-N1685, M1-Q1684, M1-L1683, M1-S1682, M1-A1681, M1-T1680,M1-N1679, M1-R1678, M1-Q1677, M1-R1676, M1-D1675, M1-L1674, M1-K1673,M1-D1672, M1-S1671, M1-I1670, M1-S1669, M1-F1668, M1-S1667, M1-K1666,M1-R1665, M1-T1664, M1-H1663, M1-A1662, M1-Y1661, M1-P1660, M1-A1659,M1-S1658, M1-P1657, M1-E1656, M1-E1655, M1-A1654, M1-S1653, M1-Y1652,M1-S1651, M1-N1650, M1-A1649, M1-R1648, M1-E1647, M1-I1646, M1-K1645,M1-P1644, M1-V1643, M1-T1642, M1-I1641, M1-N1640, M1-N1639, M1-S1638,M1-L1637, M1-T1636, M1-R1635, M1-E1634, M1-S1633, M1-N1632, M1-D1631,M1-G1630, M1-E1629, M1-Q1628, M1-S1627, M1-S1626, M1-I1625, M1-A1624,M1-I1623, M1-T1622, M1-A1621, M1-R1620, M1-R1619, M1-G1618, M1-K1617,M1-A1616, M1-E1615, M1-N1614, M1-E1613, M1-E1612, M1-S1611, M1-D1610,M1-S1609, M1-S1608, M1-P1607, M1-H1606, M1-S1605, M1-L1604, M1-E1603,M1-A1602, M1-E1601, M1-R1600, M1-E1599, M1-P1598, M1-H1597, M1-C1596,M1-C1595, M1-T1594, M1-L1593, M1-D1592, M1-E1591, M1-V1590, M1-K1589,M1-D1588, M1-G1587, M1-L1586, M1-G1585, M1-G1584, M1-P1583, M1-F1582,M1-A1581, M1-A1580, M1-R1579, M1-D1578, M1-A1577, M1-I1576, M1-A1575,M1-Q1574, M1-P1573, M1-A1572, M1-N1571, M1-V1570, M1-C1569, M1-R1568,M1-T1567, M1-D1566, M1-I1565, M1-C1564, M1-D1563, M1-T1562, M1-I1561,M1-S1560, M1-T1559, M1-Y1558, M1-E1557, M1-A1556, M1-T1555, M1-K1554,M1-V1553, M1-P1552, M1-V1551, M1-G1550, M1-F1549, M1-N1548, M1-A1547,M1-Y1546, M1-Y1545, M1-S1544, M1-R1543, M1-S1542, M1-P1541, M1-S1540,M1-F1539, M1-M1538, M1-F1537, M1-S1536, M1-H1535, M1-S1534, M1-K1533,M1-V1532, M1-I1531, M1-P1530, M1-A1529, M1-E1528, M1-E1527, M1-L1526,M1-L1525, M1-F1524, M1-P1523, M1-T1522, M1-T1521, M1-A1520, M1-L1519,M1-Y1518, M1-R1517, M1-S1516, M1-S1515, M1-K1514, M1-S1513, M1-R1512,M1-E1511, M1-I1510, M1-T1509, M1-H1508, M1-Y1507, M1-M1506, M1-P1505,M1-P1504, M1-E1503, M1-S1502, M1-D1501, M1-W1500, M1-P1499, M1-N1498,M1-Q1497, M1-C1496, M1-E1495, M1-P1494, M1-L1493, M1-H1492, M1-T1491,M1-Y1490, M1-D1489, M1-S1488, M1-S1487, M1-F1486, M1-S1485, M1-R1484,M1-T1483, M1-D1482, M1-M1481, M1-S1480, M1-T1479, M1-I1478, M1-D1477,M1-E1476, M1-F1475, M1-D1474, M1-I1473, M1-S1472, M1-R1471, M1-S1470,M1-P1469, M1-P1468, M1-R1467, M1-D1466, M1-T1465, M1-P1464, M1-A1463,M1-L1462, M1-T1461, M1-A1460, M1-Y1459, M1-A1458, M1-S1457, M1-S1456,M1-S1455, M1-P1454, M1-A1453, M1-T1452, M1-S1451, M1-P1450, M1-V1449,M1-P1448, M1-T1447, M1-S1446, M1-F1445, M1-S1444, M1-P1443, M1-E1442,M1-G1441, M1-L1440, M1-G1439, M1-L1438, M1-I1437, M1-N1436, M1-V1435,M1-S1434, M1-N1433, M1-D1432, M1-L1431, M1-P1430, M1-D1429, M1-I1428,M1-D1427, M1-C1426, M1-H1425, M1-L1424, M1-E1423, M1-D1422, M1-M1421,M1-A1420, M1-S1419, M1-V1418, M1-Y1417, M1-I1416, M1-D1415, M1-I1414,M1-C1413, M1-S1412, M1-S1411, M1-P1410, M1-R1409, M1-R1408, M1-S1407,M1-D1406, M1-P1405, M1-V1404, M1-I1403, M1-A1402, M1-L1401, M1-T1400,M1-N1399, M1-A1398, M1-P1397, M1-A1396, M1-A1395, M1-P1394, M1-A1393,M1-K1392, M1-P1391, M1-E1390, M1-K1389, M1-A1388, M1-V1387, M1-S1386,M1-H1385, M1-S1384, M1-S1383, M1-T1382, M1-A1381, M1-R1380, M1-H1379,M1-L1378, M1-S1377, M1-L1376, M1-S1375, M1-R1374, M1-E1373, M1-K1372,M1-F1371, M1-I1370, M1-S1369, M1-E1368, M1-L1367, M1-K1366, M1-E1365,M1-I1364, M1-G1363, M1-G1362, M1-K1361, M1-D1360, M1-K1359, M1-M1358,M1-N1357, M1-V1356, M1-S1355, M1-Y1354, M1-F1353, M1-S1352, M1-H1351,M1-S1350, M1-R1349, M1-M1348, M1-R1347, M1-P1346, M1-M1345, M1-L1344,M1-T1343, M1-P1342, M1-S1341, M1-T1340, M1-P1339, M1-S1338, M1-M1337,M1-T1336, M1-E1335, M1-E1334, M1-G1333, M1-A1332, M1-P1331, M1-D1330,M1-I1329, M1-S1328, M1-E1327, M1-Q1326, M1-L1325, M1-K1324, M1-F1323,M1-T1322, M1-N1321, M1-G1320, M1-E1319, M1-Q1318, M1-S1317, M1-N1316,M1-F1315, M1-S1314, M1-S1313, M1-Q1312, M1-R1311, M1-V1310, M1-I1309,M1-Y1308, M1-A1307, M1-A1306, M1-D1305, M1-T1304, M1-C1303, M1-D1302,M1-S1301, M1-S1300, M1-T1299, M1-R1298, M1-S1297, M1-R1296, M1-I1295,M1-K1294, M1-N1293, M1-S1292, M1-E1291, M1-A1290, M1-R1289, M1-E1288,M1-L1287, M1-G1286, M1-T1285, M1-L1284, M1-R1283, M1-E1282, M1-L1281,M1-A1280, M1-T1279, M1-A1278, M1-M1277, M1-R1276, M1-G1275, M1-I1274,M1-L1273, M1-D1272, M1-E1271, M1-L1270, M1-Q1269, M1-A1268, M1-L1267,M1-R1266, M1-I1265, M1-D1264, M1-V1263, M1-T1262, M1-Q1261, M1-L1260,M1-S1259, M1-A1258, M1-K1257, M1-M1256, M1-S1255, M1-H1254, M1-E1253,M1-R1252, M1-E1251, M1-N1250, M1-V1249, M1-E1248, M1-E1247, M1-L1246,M1-R1245, M1-M1244, M1-S1243, M1-M1242, M1-N1241, M1-E1240, M1-V1239,M1-R1238, M1-E1237, M1-S1236, M1-T1235, M1-V1234, M1-R1233, M1-I1232,M1-R1231, M1-E1230, M1-D1229, M1-N1228, M1-S1227, M1-S1226, M1-N1225,M1-F1224, M1-R1223, M1-D1222, M1-D1221, M1-K1220, M1-E1219, M1-R1218,M1-F1217, M1-Y1216, M1-E1215, M1-E1214, M1-I1213, M1-C1212, M1-Q1211,M1-E1210, M1-E1209, M1-F1208, M1-D1207, M1-H1206, M1-V1205, M1-K1204,M1-K1203, M1-L1202, M1-E1201, M1-D1200, M1-D1199, M1-T1198, M1-I1197,M1-F1196, M1-L1195, M1-K1194, M1-L1193, M1-G1192, M1-Y1191, M1-D1190,M1-R1189, M1-E1188, M1-D1187, M1-P1186, M1-D1185, M1-S1184, M1-E1183,M1-H1182, M1-K1181, M1-R1180, M1-W1179, M1-R1178, M1-C1177, M1-C1176,M1-L1175, M1-H1174, M1-Q1173, M1-F1172, M1-I1171, M1-M1170, M1-T1169,M1-M1168, M1-H1167, M1-S1166, M1-F1165, M1-I1164, M1-I1163, M1-L1162,M1-P1161, M1-P1160, M1-P1159, M1-L1158, M1-V1157, M1-P1156, M1-R1155,M1-E1154, M1-H1153, M1-F1152, M1-T1151, M1-M1150, M1-I1149, M1-L1148,M1-Q1147, M1-Y1146, M1-R1145, M1-Q1144, M1-F1143, M1-K1142, M1-W1141,M1-V1140, M1-Q1139, M1-N1138, M1-S1137, M1-I1136, M1-S1135, M1-K1134,M1-V1133, M1-E1132, M1-F1131, M1-F1130, M1-T1129, M1-N1128, M1-N1127,M1-F1126, M1-V1125, M1-A1124, M1-I1123, M1-L1122, M1-L1121, M1-N1120,M1-V1119, M1-L1118, M1-L1117, M1-I1116, M1-N115, M1-A1114, M1-V1113,M1-L1112, M1-L1111, M1-Y1110, M1-C1109, M1-A1108, M1-M1107, M1-I1106,M1-A1105, M1-P1104, M1-V1103, M1-I1102, M1-W1101, M1-A1100, M1-G1099,M1-T1098, M1-K1097, M1-C1096, M1-P1095, M1-P1094, M1-L1093, M1-Q1092,M1-I1091, M1-I1090, M1-K1089, M1-G1088, M1-D1087, M1-E1086, M1-R1085,M1-T1084, M1-E1083, M1-N1082, M1-Q1081, M1-G1080, M1-C1079, M1-P1078,M1-A1077, M1-S1076, M1-K1075, M1-P1074, M1-T1073, M1-H1072, M1-S1071,M1-D1070, M1-Y1069, M1-V1068, M1-Q1067, M1-K1066, M1-R1065, M1-D1064,M1-I1063, M1-Q1062, M1-D1061, M1-A1060, M1-F1059, M1-V1058, M1-E1057,M1-G1056, M1-Y1055, M1-I1054, M1-M1053, M1-W1052, M1-Y1051, M1-P1050,M1-M1049, M1-Y1048, M1-F1047, M1-I1046, M1-N1045, M1-K1044, M1-A1043,M1-L1042, M1-K1041, M1-W1040, M1-S1039, M1-P1038, M1-E1037, M1-E1036,M1-N1035, M1-P1034, M1-F1033, M1-L1032, M1-I1031, M1-A1030, M1-Q1029,M1-R1028, M1-A1027, M1-V1026, M1-G1025, M1-F1024, M1-S1023, M1-M1022,M1-L1021, M1-V1020, M1-V1019, M1-L1018, M1-M1017, M1-I1016, M1-I1015,M1-V1014, M1-F1013, M1-Y1012, M1-M1011, M1-M1010, M1-D1009, M1-I1008,M1-M1007, M1-M1006, M1-K1005, M1-G1004, M1-I1003, M1-M1002, M1-M1001,M1-V1000, M1-Y999, M1-P998, M1-G997, M1-L996, M1-Y995, M1-K994, M1-N993,M1-V992, M1-G991, M1-F990, M1-I989, M1-D988, M1-L987, M1-L986, M1-R985,M1-I984, M1-Y983, M1-W982, M1-Y981, M1-I980, M1-I979, M1-N978, M1-V977,M1-C976, M1-Y975, M1-I974, M1-V973, M1-R972, M1-G971, M1-D970, M1-S969,M1-R968, M1-F967, M1-P966, M1-Q965, M1-D964, M1-Q963, M1-L962, M1-R961,M1-L960, M1-I959, M1-M958, M1-G957, M1-V956, M1-S955, M1-F954, M1-L953,M1-L952, M1-I951, M1-A950, M1-I949, M1-L948, M1-D947, M1-T946, M1-V945,M1-N944, M1-W943, M1-Y942, M1-E941, M1-Q940, M1-L939, M1-W938, M1-V937,M1-K936, M1-V935, M1-K934, M1-Q933, M1-L932, M1-L931, M1-K930, M1-G929,M1-P928, M1-E927, M1-S926, M1-M925, M1-L924, M1-I923, M1-E922, M1-R921,M1-M920, M1-K919, M1-E918, M1-I917, M1-G916, M1-L915, M1-T914, M1-F913,M1-I912, M1-Y911, M1-S910, M1-I909, M1-V908, M1-I907, M1-W906, M1-E905,M1-Q904, M1-T903, M1-S902, M1-P901, M1-W900, M1-R899, M1-E898, M1-M897,M1-K896, M1-V895, M1-L894, M1-V893, M1-I892, M1-Y891, M1-N890, M1-F889,M1-L888, M1-M887, M1-L886, M1-Y885, M1-G884, M1-I883, M1-Y882, M1-A881,M1-L880, M1-T879, M1-Y878, M1-F877, M1-W876, M1-F875, M1-K874, M1-V873,M1-I872, M1-P871, M1-A870, M1-N869, M1-Y868, M1-F867, M1-E866, M1-Y865,M1-I864, M1-K863, M1-R862, M1-G861, M1-L860, M1-P859, M1-I858, M1-L857,M1-R856, M1-H855, M1-K854, M1-S853, M1-Q852, M1-V851, M1-E850, M1-E849,M1-E848, M1-D847, M1-K846, M1-K845, M1-R844, M1-S843, M1-S842, M1-E841,M1-G840, M1-N839, M1-N838, M1-R837, M1-G836, M1-L835, M1-M834, M1-A833,M1-T832, M1-L831, M1-E830, M1-M829, M1-D828, M1-E827, M1-E826, M1-E825,M1-K824, M1-E823, M1-K822, M1-T821, M1-P820, M1-K819, M1-E818, M1-P817,M1-E816, M1-E815, M1-A814, M1-E813, M1-K812, M1-E811, M1-Q810, M1-L809,M1-H808, M1-I807, M1-E806, M1-Q805, M1-A804, M1-Q803, M1-S802, M1-M801,M1-Y800, M1-P799, M1-M798, M1-D797, M1-D796, M1-K795, M1-N794, M1-K793,M1-F792, M1-E791, M1-L790, M1-S789, M1-L788, M1-I787, M1-S786, M1-P785,M1-P784, M1-L783, M1-L782, M1-I781, M1-G780, M1-L779, M1-I778, M1-V777,M1-K776, M1-L775, M1-G774, M1-S773, M1-N772, M1-K771, M1-R770, M1-M769,M1-R768, M1-L767, M1-R766, M1-G765, M1-M764, M1-W763, M1-M762, M1-D761,M1-T760, M1-L759, M1-L758, M1-M757, M1-Q756, M1-S755, M1-C754, M1-T753,M1-H752, M1-A751, M1-I750, M1-F749, M1-D748, M1-R747, M1-H746, M1-K745,M1-A744, M1-A743, M1-V742, M1-A741, M1-L740, M1-Q739, M1-L738, M1-C737,M1-T736, M1-A735, M1-N734, M1-S733, M1-W732, M1-N731, M1-K730, M1-L729,M1-E728, M1-Y727, M1-T726, M1-L725, M1-L724, M1-K723, M1-M722, M1-A721,M1-L720, M1-Q719, M1-E718, M1-D717, M1-Q716, M1-K715, M1-Y714, M1-S713,M1-Q712, M1-D711, M1-L710, M1-L709, M1-E708, M1-V707, M1-A706, M1-L705,M1-Q704, M1-G703, M1-F702, M1-D701, M1-R700, M1-S699, M1-N698, M1-H697,M1-N696, M1-L695, M1-E694, M1-Q693, M1-S692, M1-I691, M1-D690, M1-D689,M1-V688, M1-M687, M1-D686, M1-N685, M1-E684, M1-S683, M1-A682, M1-E681,M1-H680, M1-A679, M1-M678, M1-A677, M1-K676, M1-C675, M1-L674, M1-K673,M1-C672, M1-A671, M1-V670, M1-L669, M1-A668, M1-K667, M1-A666, M1-M665,M1-A664, M1-E663, M1-E662, M1-G661, M1-H660, M1-Q659, M1-W658, M1-F657,M1-F656, M1-L655, M1-A654, M1-M653, M1-K652, M1-Q651, M1-R650, M1-K649,M1-M648, M1-L647, M1-V646, M1-A645, M1-W644, M1-V643, M1-M642, M1-L641,M1-E640, M1-H639, M1-F638, M1-P637, M1-F636, M1-P635, M1-F634, M1-H633,M1-N632, M1-I631, M1-E630, M1-P629, M1-D628, M1-D627, M1-L626, M1-D625,M1-I624, M1-D623, M1-V622, M1-E621, M1-E620, M1-E619, M1-R618, M1-K617,M1-K616, M1-T615, M1-T614, M1-K613, M1-R612, M1-G611, M1-R610, M1-R609,M1-L608, M1-P607, M1-I606, M1-D605, M1-D604, M1-E603, M1-M602, M1-G601,M1-L600, M1-L599, M1-K598, M1-L597, M1-A596, M1-K595, M1-P594, M1-R593,M1-K592, M1-P591, M1-G590, M1-F589, M1-L588, M1-N587, M1-H586, M1-Y585,M1-L584, M1-T583, M1-R582, M1-F581, M1-R580, M1-K579, M1-R578, M1-T577,M1-Y576, M1-N575, M1-C574, M1-R573, M1-Y572, M1-A571, M1-G570, M1-G569,M1-M568, M1-L567, M1-Y566, M1-E565, M1-I564, M1-V563, M1-L562, M1-G561,M1-I560, M1-D559, M1-I558, M1-L557, M1-S556, M1-I555, M1-R554, M1-Y553,M1-D552, M1-P551, M1-P550, M1-L549, M1-N548, M1-G547, M1-K546, M1-K545,M1-V544, M1-D543, M1-R542, M1-V541, M1-L540, M1-H539, M1-Y538, M1-L537,M1-T536, M1-N535, M1-S534, M1-P533, M1-G532, M1-H531, M1-R530, M1-T529,M1-N528, M1-Y527, M1-L526, M1-E525, M1-E524, M1-L523, M1-R522, M1-S521,M1-I520, M1-T519, M1-L518, M1-F517, M1-R516, M1-H515, M1-M514, M1-S513,M1-V512, M1-G511, M1-N510, M1-E509, M1-I508, M1-L507, M1-L506, M1-K505,M1-V504, M1-F503, M1-D502, M1-V501, M1-R500, M1-D499, M1-L498, M1-V497,M1-L496, M1-A495, M1-D494, M1-L493, M1-M492, M1-A491, M1-Q490, M1-E489,M1-LA88, M1-S487, M1-G486, M1-V485, M1-P484, M1-W483, M1-Q482, M1-Q481,M1-G480, M1-Y479, M1-I478, M1-F477, M1-I476, M1-Q475, M1-S474, M1-R473,M1-A472, M1-I471, M1-D470, M1-V469, M1-R468, M1-N467, M1-W466, M1-A465,M1-L464, M1-A463, M1-L462, M1-S461, M1-L460, M1-Q459, M1-D458, M1-P457,M1-A456, M1-S455, M1-A454, M1-N453, M1-A452, M1-G451, M1-K450, M1-L449,M1-L448, M1-A447, M1-T446, M1-L445, M1-I444, M1-A443, M1-L442, M1-D441,M1-I440, M1-D439, M1-Q438, M1-H437, M1-G436, M1-E435, M1-S434, M1-G433,M1-M432, M1-R431, M1-F430, M1-V429, M1-T428, M1-I427, M1-L426, M1-E425,M1-K424, M1-K423, M1-K422, M1-M421, M1-C420, M1-E419, M1-M418, M1-L417,M1-I416, M1-I415, M1-F414, M1-L413, M1-H412, M1-Q411, M1-A410, M1-Q409,M1-T408, M1-R407, M1-T406, M1-Y405, M1-T404, M1-F403, M1-T402, M1-K401,M1-Q400, M1-I399, M1-T398, M1-V397, M1-L396, M1-L395, M1-Q394, M1-D393,M1-R392, M1-L391, M1-S390, M1-E389, M1-N388, M1-I387, M1-L386, M1-G385,M1-G384, M1-E383, M1-E382, M1-S381, M1-Y380, M1-K379, M1-H378, M1-G377,M1-F376, M1-A375, M1-L374, M1-I373, M1-D372, M1-S371, M1-A370, M1-R369,M1-G368, M1-S367, M1-G366, M1-D365, M1-C364, M1-V363, M1-V362, M1-V361,M1-P360, M1-V359, M1-P358, M1-P357, M1-T356, M1-D355, M1-R354, M1-L353,M1-Y352, M1-E351, M1-L350, M1-V349, M1-I348, M1-S347, M1-I346, M1-V345,M1-N344, M1-P343, M1-G342, M1-G341, M1-E340, M1-V339, M1-I338, M1-L337,M1-A336, M1-V335, M1-V334, M1-P333, M1-V332, M1-G331, M1-Q330, M1-G329,M1-I328, M1-R327, M1-T326, M1-N325, M1-I324, M1-K323, M1-Q322, M1-L321,M1-S320, M1-I319, M1-H318, M1-K317, M1-E316, M1-L315, M1-Q314, M1-R313,M1-R312, M1-L311, M1-K310, M1-V309, M1-E308, M1-A307, M1-G306, M1-Y305,M1-K304, M1-G303, M1-T302, M1-T301, M1-G300, M1-N299, M1-D298, M1-A297,M1-L296, M1-I295, M1-F294, M1-H293, M1-S292, M1-H291, M1-M290, M1-S289,M1-N288, M1-L287, M1-V286, M1-T285, M1-L284, M1-K283, M1-S282, M1-M281,M1-P280, M1-N279, M1-S278, M1-M277, M1-T276, M1-Q275, M1-Y274, M1-P273,M1-R272, M1-V271, M1-V270, M1-D269, M1-R268, M1-G267, M1-I266, M1-L265,M1-D264, M1-E263, M1-Q262, M1-N261, M1-E260, M1-V259, M1-I258, M1-G257,M1-W256, M1-P255, M1-A254, M1-I253, M1-G252, M1-I251, M1-T250, M1-C249,M1-I248, M1-K247, M1-G246, M1-R245, M1-S244, M1-K243, M1-S242, M1-A241,M1-H240, M1-D239, M1-K238, M1-L237, M1-A236, M1-D235, M1-G234, M1-V233,M1-H232, M1-R231, M1-I230, M1-V229, M1-G228, M1-T227, M1-N226, M1-V225,M1-G224, M1-G223, M1-T222, M1-F221, M1-I220, M1-W219, M1-A218, M1-G217,M1-T216, M1-T215, M1-M214, M1-A213, M1-A212, M1-K211, M1-I210, M1-L209,M1-G208, M1-K207, M1-G206, M1-F205, M1-V204, M1-Q203, M1-K202, M1-L201,M1-K200, M1-P199, M1-Q198, M1-L197, M1-E196, M1-F195, M1-N194, M1-Q193,M1-L192, M1-G191, M1-G190, M1-H189, M1-V188, M1-S187, M1-I186, M1-L185,M1-L184, M1-K183, M1-P182, M1-L181, M1-E180, M1-L179, M1-Q178, M1-W177,M1-E176, M1-K175, M1-T174, M1-M173, M1-L172, M1-H171, M1-L170, M1-L169,M1-L168, M1-D167, M1-P166, M1-K165, M1-T164, M1-D163, M1-F162, M1-S161,M1-V160, M1-R159, M1-V158, M1-Y157, M1-M156, M1-A155, M1-K154, M1-N153,M1-S152, M1-H151, M1-G150, M1-G149, M1-G148, M1-Q147, M1-F146, M1-E145,M1-I144, M1-T143, M1-G142, M1-F141, M1-A140, M1-D139, M1-T138, M1-P137,M1-S136, M1-L135, M1-Q134, M1-T133, M1-H132, M1-K131, M1-S130, M1-I129,M1-S128, M1-W127, M1-K126, M1-E125, M1-S124, M1-Q123, M1-I122, M1-D121,M1-N120, M1-R119, M1-S118, M1-L117, M1-R116, M1-S115, M1-E114, M1-N113,M1-K112, M1-E111, M1-N110, M1-Q109, M1-L108, M1-V107, M1-S106, M1-I105,M1-S104, M1-P103, M1-T102, M1-L101, M1-G100, M1-V99, M1-H98, M1-Q97,M1-G96, M1-I95, M1-L94, M1-R93, M1-G92, M1-C91, M1-C90, M1-C89, M1-R88,M1-H87, M1-P86, M1-D85, M1-K84, M1-T83, M1-S82, M1-P81, M1-I80, M1-I79,M1-H78, M1-V77, M1-C76, M1-E75, M1-R74, M1-K73, M1-Y72, M1-F71, M1-A70,M1-R69, M1-E68, M1-I67, M1-W66, M1-S65, M1-K64, M1-Q63, M1-A62, M1-P61,M1-T60, M1-Q59, M1-Q58, M1-S57, M1-P56, M1-L55, M1-G54, M1-S53, M1-R52,M1-K51, M1-T50, M1-S49, M1-G48, M1-Q47, M1-K46, M1-S45, M1-S44, M1-L43,M1-R42, M1-K41, M1-W40, M1-S39, M1-E38, M1-A37, M1-F36, M1-R35, M1-G34,M1-R33, M1-S32, M1-A31, M1-S30, M1-R29, M1-S28, M1-R27, M1-R26, M1-R25,M1-S24, M1-E23, M1-A22, M1-N21, M1-D20, M1-E19, M1-R18, M1-D17, M1-S16,M1-C15, M1-G14, M1-R13, M1-E12, M1-M11, M1-E10, M1-A9, M1-A8, and/orM1-D7 of SEQ ID NO:6. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal LTRPC3i deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

[0379] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3i polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3i polypeptide deletions) of SEQ ID NO:6.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3i (SEQ ID NO:6), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3i (SEQ ID NO:6).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0380] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:5 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5223 ofSEQ ID NO:5, b is an integer between 15 to 5237, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:5,and where b is greater than or equal to a+14.

[0381] Features of the Polypeptide Encoded by Gene No:4

[0382] The polypeptide of this gene provided as SEQ ID NO:8 (FIGS.4A-F), encoded by the polynucleotide sequence according to SEQ ID NO:7(FIGS. 4A-F), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3j, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3jpolypeptide with this protein is provided in FIGS. 7A-G.

[0383] The LTRPC3j polypeptide was determined to share 65.4% identityand 73.3% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0384] The LTRPC3j protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0385] Specifically, the LTRPC3j (SEQ ID NO:8) polypeptide represents anovel variant of the LTRPC3d (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:19; and also co-pending U.S. Ser. No. 10/405,793,filed Mar. 28, 2003; which are hereby incorporated by reference hereinin their entirety) polypeptide. The LTRPC3j represents a novel member ofthe TRPM subfamily.

[0386] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0387] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art, particularly LTRPC3d, and LTRPC3.

[0388] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3j polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 4A-F. The transmembrane domains arelocated from about amino acid 775 to about amino acid 792 (TM1), fromabout amino acid 872 to about amino acid 888 (TM2), from about aminoacid 945 to about amino acid 958 (TM3), from about amino acid 972 toabout amino acid 988 (TM4), from about amino acid 1006 to about aminoacid 1023 (TM5), and/or from about amino acid 1115 to about amino acid1125 (TM6) of SEQ ID NO:8. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0389] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:64), IVKFWFYTLAYIGYLMLF (SEQ ID NO:65),VTDLIAILLFSVGM (SEQ ID NO:66), RVIYCVNIIYWYIRLLDI (SEQ ID NO:67),MMIDMMYFVIIMLVVLMS (SEQ ID NO:68), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:69). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3jtransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0390] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3j, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3j polypeptide,particularly for the development of antibodies specific to theseregions.

[0391] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:70),NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:71),ILRLQDQPFRSDG (SEQ ID NO:72), FGVNKYLGPYVMMIGK (SEQ ID NO:73), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDRKQVYDSHTPKSAPCGQNETREDGKIIQLPPCKTGAWIVP (SEQ ID NO:74). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of the LTRPC3j inter-transmembrane polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0392] The LTRPC3j polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 364, 420, 574, 662, 727, 744,966, 1057, 1074, 1087, 1190, 1281, and 1547 of SEQ ID NO:8 (FIGS. 4A-F).Conservation of cysteines at key amino acid residues is indicative ofconserved structural features, which may correlate with conservation ofprotein function and/or activity.

[0393] In confirmation of the LTRPC3j representing a member of thetransient receptor channel family, the LTRPC3j polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1118 to about amino acid 1123 of SEQ ID NO:8. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0394] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:75).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3j TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0395] In further confirmation of the LTRPC3j representing a member ofthe transient receptor channel family, the LTRPC3j polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 893 to about amino acid 1104 of SEQ ID NO:8.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminusof the above referenced polypeptide.

[0396] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFILGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:76).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3j ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0397] The LTRPC3j polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1208 to aboutamino acid 1263 of SEQ ID NO:8. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0398] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:77).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3j coiled-coil domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0399] LTRPC3j polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3j by identifying mutations in the LTRPC3j gene using LTRPC3jsequences as probes or by determining LTRPC3j protein or mRNA expressionlevels. LTRPC3j polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3j peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3j.

[0400] Since the LTRPC3j polypeptide represents an N-terminally extendedvariant of the LTRPC3d polypeptide (see polypeptide alignment providedin FIG. 11A-I), and since the LTRPC3d (SEQ ID NO:19) polypeptiderepesents a splice variant of the LTRPC3 polypeptide (SEQ ID NO:15), theLTRPC3j (SEQ ID NO:8) polypeptide of the present invention is expectedto have the same or similar function as the LTRPC3 polypeptide, and inparticular, the LTRPC3d polypeptide. Specifically, the LTRPC3jpolypeptide is expected to localize to the cell membrane.

[0401] Moreover, the LTRPC3j polypeptide is expected to also localize inor near the plasmalemmal compartment.

[0402] Moreover, the LTRPC3j polypeptide is also expected to be aconstitutively active channel capable of mediating Ca²⁺ influx.

[0403] Moreover, the LTRPC3j polypeptide is also expected to mediateCa²⁺ entry and that LTRPC3j-mediated Ca²⁺ entry is expected to bepotentiated by store-depletion.

[0404] Moreover, the LTRPC3j polypeptide is also expected to mediate aCa²⁺ entry pathway that is distinct from the endogenous Ca²⁺ entrypathways present in HEK 293 cells.

[0405] Moreover, the LTRPC3j polypeptide of the present invention isexpected to share the same or similar expression profile as that of theLTRPC3 polypeptide (SEQ ID NO:15).

[0406] Moreover, the LTRPC3j polypeptide is expected to share the sameor similar differential expression patterns observed for the LTRPC3variant.

[0407] Moreover, the LTRCP3g polypeptide is expected to be able tomodulate the FEN1 DNA base-excision repair/proliferation modulatingprotein to the same or similar extent as the LTRPC3 polypeptide.

[0408] Moreover, the LTRPC3j polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein, such as LTRPC3 (SEQ ID NO:15), andparticularly as LTRPC3b (SEQ ID NO:17).

[0409] Moreover, the LTRPC3j polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein, such as the LTRPC3 variant (SEQ IDNO:15), and particularly as LTRPC3d (SEQ ID NO:19).

[0410] In preferred embodiments, LTRPC3j polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0411] In preferred embodiments, LTRPC3j polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0412] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0413] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0414] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3j to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3j activity or a compound that increased LTRPC3j message levelswould be a desired invention for cancer therapy.

[0415] In preferred embodiments, LTRPC3j polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0416] Moreover, LTRPC3j polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0417] In preferred embodiments, antagonists directed against LTRPC3jare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0418] Moreover, antagonists directed against LTRPC3j are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0419] In preferred embodiments, agonists directed against LTRPC3j areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0420] Moreover, agonists directed against LTRPC3j are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0421] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3jpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0422] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0423] Thus, the LTRPC3j polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0424] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3jpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0425] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3jpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0426] In preferred embodiments, LTRPC3j polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3jpolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0427] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3j polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0428] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0429] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0430] Thus, the LTRPC3j polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0431] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0432] In preferred embodiments, LTRPC3j polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0433] In more preferred embodiments, LTRPC3j polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0434] LTRPC3j polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3jpolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0435] The LTRPC3j gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3j can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0436] Moreover, given the selective expression in kidney, LTRPC3j couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3j chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3j is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3j inthe disease. Therefore, it is possible that LTRPC3j may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0437] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3j may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3j may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0438] In addition, LTRPC3j polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0439] The LTRPC3j polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3j polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0440] In addition, antagonists of the LTRPC3j polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0441] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin 1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3j polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3j, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0442] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3j gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0443] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3j, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3j and assessing their ability to grow wouldprovide convincing evidence the LTRPC3j polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0444] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0445] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0446] In the case of LTRPC3j transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0447] In preferred embodiments, the following N-terminal LTRPC3jdeletion polypeptides are encompassed by the present invention:M1-T1699, G2-T1699, K3-T1699, K4-T1699, W5-T1699, R6-T1699, D7-T1699,A8-T1699, A9-T1699, E10-T1699, M11-T1699, E12-T1699, R13-T1699,G14-T1699, C15-T1699, S16-T1699, D17-T1699, R18-T1699, E19-T1699,D20-T1699, N21-T1699, A22-T1699, E23-T1699, S24-T1699, R25-T1699,R26-T1699, R27-T1699, S28-T1699, R29-T1699, S30-T1699, A31-T1699,S32-T1699, R33-T1699, G34-T1699, R35-T1699, F36-T1699, A37-T1699,E38-T1699, S39-T1699, W40-T1699, K41-T1699, R42-T1699, L43-T1699,S44-T1699, S45-T1699, K46-T1699, Q47-T1699, G48-T1699, S49-T1699,T50-T1699, K51-T1699, R52-T1699, S53-T1699, G54-T1699, L55-T1699,P56-T1699, S57-T1699, Q58-T1699, Q59-T1699, T60-T1699, P61-T1699,A62-T1699, Q63-T1699, K64-T1699, S65-T1699, W66-T1699, I67-T1699,E68-T1699, R69-T1699, A70-T1699, F71-T1699, Y72-T1699, K73-T1699,R74-T1699, E75-T1699, C76-T1699, V77-T1699, H78-T1699, I79-T1699,I80-T1699, P81-T1699, S82-T1699, T83-T1699, K84-T1699, D85-T1699,P86-T1699, H87-T1699, R88-T1699, C89-T1699, C90-T1699, C91-T1699,G92-T1699, R93-T1699, L94-T1699, I95-T1699, G96-T1699, Q97-T1699,H98-T1699, V99-T1699, G100-T1699, L10-T1699, T102-T1699, P103-T1699,S104-T1699, I105-T1699, S106-T1699, V107-T1699, L108-T1699, Q109-T1699,N110-T1699, E111-T1699, K112-T1699, N113-T1699, E114-T1699, S115-T1699,R116-T1699, L117-T1699, S118-T1699, R119-T1699, N120-T1699, D121-T1699,I122-T1699, Q123-T1699, S124-T1699, E125-T1699, K126-T1699, W127-T1699,S128-T1699, I129-T1699, S130-T1699, K131-T1699, H132-T1699, T133-T1699,Q134-T1699, L135-T1699, S136-T1699, P137-T1699, T138-T1699, D139-T1699,A140-T1699, F141-T1699, G142-T1699, T143-T1699, I144-T1699, E145-T1699,F146-T1699, Q147-T1699, G148-T1699, G149-T1699, G150-T1699, H151-T1699,S152-T1699, N153-T1699, K154-T1699, and/or A155-T1699 of SEQ ID NO:8.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3j deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0448] In preferred embodiments, the following C-terminal LTRPC3jdeletion polypeptides are encompassed by the present invention:M1-T1699, M1-H1698, M1-K1697, M1-S1696, M1-E1695, M1-F1694, M1-S1693,M1-Q1692, M1-F1691, M1-A1690, M1-S1689, M1-T1688, M1-R1687, M1-S1686,M1-L1685, M1-R1684, M1-R1683, M1-M1682, M1-S1681, M1-L1680, M1-S1679,M1-D1678, M1-G1677, M1-R1676, M1-G1675, M1-E1674, M1-P1673, M1-K1672,M1-S1671, M1-S1670, M1-K1669, M1-S1668, M1-R1667, M1-Q1666, M1-F1665,M1-P1664, M1-N1663, M1-Q1662, M1-L1661, M1-S1660, M1-A1659, M1-T1658,M1-N1657, M1-R1656, M1-Q1655, M1-R1654, M1-D1653, M1-L1652, M1-K1651,M1-D1650, M1-S1649, M1-I1648, M1-S1647, M1-F1646, M1-S1645, M1-K1644,M1-R1643, M1-T1642, M1-H1641, M1-A1640, M1-Y1639, M1-P1638, M1-A1637,M1-S1636, M1-P1635, M1-E1634, M1-E1633, M1-A1632, M1-S1631, M1-Y1630,M1-S1629, M1-N1628, M1-A1627, M1-R1626, M1-E1625, M1-I1624, M1-K1623,M1-P1622, M1-V1621, M1-T1620, M1-I1619, M1-N1618, M1-N1617, M1-S1616,M1-L1615, M1-T1614, M1-R1613, M1-E1612, M1-S1611, M1-N1610, M1-D1609,M1-G1608, M1-E1607, M1-Q1606, M1-S1605, M1-S1604, M1-I1603, M1-A1602,M1-I1601, M1-T1600, M1-A1599, M1-R1598, M1-R1597, M1-G1596, M1-K1595,M1-A1594, M1-E1593, M1-N1592, M1-E1591, M1-E1590, M1-S1589, M1-D1588,M1-S1587, M1-S1586, M1-P1585, M1-H1584, M1-S1583, M1-L1582, M1-E1581,M1-A1580, M1-E1579, M1-R1578, M1-E1577, M1-P1576, M1-H1575, M1-C1574,M1-C1573, M1-T1572, M1-L1571, M1-D1570, M1-E1569, M1-V1568, M1-K1567,M1-D1566, M1-G1565, M1-L1564, M1-G1563, M1-G1562, M1-P1561, M1-F1560,M1-A1559, M1-A1558, M1-R1557, M1-D1556, M1-A1555, M1-I1554, M1-A1553,M1-Q1552, M1-P1551, M1-A1550, M1-N1549, M1-V1548, M1-C1547, M1-R1546,M1-T1545, M1-D1544, M1-I1543, M1-C1542, M1-D1541, M1-T1540, M1-I1539,M1-S1538, M1-T1537, M1-Y1536, M1-E1535, M1-A1534, M1-T1533, M1-K1532,M1-V1531, M1-P1530, M1-V1529, M1-G1528, M1-F1527, M1-N1526, M1-A1525,M1-Y1524, M1-Y1523, M1-S1522, M1-R1521, M1-S1520, M1-P1519, M1-S1518,M1-F1517, M1-M1516, M1-F1515, M1-S1514, M1-H1513, M1-S1512, M1-K1511,M1-V1510, M1-I1509, M1-P1508, M1-A1507, M1-E1506, M1-E1505, M1-L1504,M1-L1503, M1-F1502, M1-P1501, M1-T1500, M1-T1499, M1-A1498, M1-L1497,M1-Y1496, M1-R1495, M1-S1494, M1-S1493, M1-K1492, M1-S1491, M1-R1490,M1-E1489, M1-I1488, M1-T1487, M1-H1486, M1-Y1485, M1-M1484, M1-P1483,M1-P1482, M1-E1481, M1-S1480, M1-D1479, M1-W1478, M1-P1477, M1-N1476,M1-Q1475, M1-C1474, M1-E1473, M1-P1472, M1-L1471, M1-H1470, M1-T1469,M1-Y1468, M1-D1467, M1-S1466, M1-S1465, M1-F1464, M1-S1463, M1-R1462,M1-T1461, M1-D1460, M1-M1459, M1-S1458, M1-T1457, M1-I1456, M1-D1455,M1-E1454, M1-F1453, M1-D1452, M1-I1451, M1-S1450, M1-R1449, M1-S1448,M1-P1447, M1-P1446, M1-R1445, M1-D1444, M1-T1443, M1-P1442, M1-A1441,M1-L1440, M1-T1439, M1-A1438, M1-Y1437, M1-A1436, M1-S1435, M1-S1434,M1-S1433, M1-P1432, M1-A1431, M1-T1430, M1-S1429, M1-P1428, M1-V1427,M1-P1426, M1-T1425, M1-S1424, M1-F1423, M1-S1422, M1-P1421, M1-E1420,M1-G1419, M1-L1418, M1-G1417, M1-L1416, M1-I1415, M1-N1414, M1-V1413,M1-S1412, M1-N1411, M1-D1410, M1-L1409, M1-P1408, M1-D1407, M1-I1406,M1-D1405, M1-C1404, M1-H1403, M1-L1402, M1-E1401, M1-D1400, M1-M1399,M1-A1398, M1-S1397, M1-V1396, M1-Y1395, M1-I1394, M1-D1393, M1-I1392,M1-C1391, M1-S1390, M1-S1389, M1-P1388, M1-R1387, M1-R1386, M1-S1385,M1-D1384, M1-P1383, M1-V1382, M1-I1381, M1-A1380, M1-L1379, M1-T1378,M1-N1377, M1-A1376, M1-P1375, M1-A1374, M1-A1373, M1-P1372, M1-A1371,M1-K1370, M1-P1369, M1-E1368, M1-K1367, M1-A1366, M1-V1365, M1-S1364,M1-H1363, M1-S1362, M1-S1361, M1-T1360, M1-A1359, M1-R1358, M1-H1357,M1-L1356, M1-S1355, M1-L1354, M1-S1353, M1-R1352, M1-E1351, M1-K1350,M1-F1349, M1-I1348, M1-S1347, M1-E1346, M1-L1345, M1-K1344, M1-E1343,M1-I1342, M1-G1341, M1-G1340, M1-K1339, M1-D1338, M1-K1337, M1-M1336,M1-N1335, M1-V1334, M1-S1333, M1-Y1332, M1-F1331, M1-S1330, M1-H1329,M1-S1328, M1-R1327, M1-M1326, M1-R1325, M1-P1324, M1-M1323, M1-L1322,M1-T1321, M1-P1320, M1-S1319, M1-T1318, M1-P1317, M1-S1316, M1-M1315,M1-T1314, M1-E1313, M1-E1312, M1-G1311, M1-A1310, M1-P1309, M1-D1308,M1-I1307, M1-S1306, M1-E1305, M1-Q1304, M1-L1303, M1-K1302, M1-F1301,M1-T1300, M1-N1299, M1-G1298, M1-E1297, M1-Q1296, M1-S1295, M1-N1294,M1-F1293, M1-S1292, M1-S1291, M1-Q1290, M1-R1289, M1-V1288, M1-I1287,M1-Y1286, M1-A1285, M1-A1284, M1-D1283, M1-T1282, M1-C1281, M1-D1280,M1-S1279, M1-S1278, M1-T1277, M1-R1276, M1-S1275, M1-R1274, M1-I1273,M1-K1272, M1-N1271, M1-S1270, M1-E1269, M1-A1268, M1-R1267, M1-E1266,M1-L1265, M1-G1264, M1-T1263, M1-L1262, M1-R1261, M1-E1260, M1-L1259,M1-A1258, M1-T1257, M1-A1256, M1-M1255, M1-R1254, M1-G1253, M1-I1252,M1-L1251, M1-D1250, M1-E1249, M1-L1248, M1-Q1247, M1-A1246, M1-L1245,M1-R1244, M1-I1243, M1-D1242, M1-V1241, M1-T1240, M1-Q1239, M1-L1238,M1-S1237, M1-A1236, M1-K1235, M1-M1234, M1-S1233, M1-H1232, M1-E1231,M1-R1230, M1-E1229, M1-N1228, M1-V1227, M1-E1226, M1-E1225, M1-L1224,M1-R1223, M1-M1222, M1-S1221, M1-M1220, M1-N1219, M1-E1218, M1-V1217,M1-R1216, M1-E1215, M1-S1214, M1-T1213, M1-V1212, M1-R1211, M1-I1210,M1-R1209, M1-E1208, M1-D1207, M1-N1206, M1-S1205, M1-S1204, M1-N1203,M1-F1202, M1-R1201, M1-D1200, M1-D1199, M1-K1198, M1-E1197, M1-R1196,M1-F1195, M1-Y1194, M1-E1193, M1-E1192, M1-I1191, M1-C1190, M1-Q1189,M1-E1188, M1-E1187, M1-F1186, M1-D1185, M1-H1184, M1-V1183, M1-K1182,M1-K1181, M1-L1180, M1-E1179, M1-D1178, M1-D1177, M1-T1176, M1-I1175,M1-F1174, M1-L1173, M1-K1172, M1-L1171, M1-G1170, M1-Y1169, M1-D1168,M1-R1167, M1-E1166, M1-D1165, M1-P1164, M1-D1163, M1-S1162, M1-E1161,M1-H1160, M1-K1159, M1-R1158, M1-W1157, M1-R1156, M1-C1155, M1-C1154,M1-L1153, M1-H1152, M1-Q1151, M1-F1150, M1-I1149, M1-M1148, M1-T1147,M1-M1146, M1-H1145, M1-S1144, M1-F1143, M1-I1142, M1-I1141, M1-L1140,M1-P1139, M1-P1138, M1-P1137, M1-L1136, M1-V1135, M1-P1134, M1-R1133,M1-E1132, M1-H1131, M1-F1130, M1-T1129, M1-M1128, M1-I1127, M1-L1126,M1-Q1125, M1-Y1124, M1-R1123, M1-Q1122, M1-F1121, M1-K1120, M1-W1119,M1-V1118, M1-Q1117, M1-N1116, M1-S1115, M1-I1114, M1-S1113, M1-K1112,M1-V1111, M1-E1110, M1-F1109, M1-F1108, M1-T1107, M1-N1106, M1-N1105,M1-F1104, M1-V1103, M1-A1102, M1-I1110, M1-L1100, M1-L1099, M1-N1098,M1-V1097, M1-L1096, M1-L1095, M1-I1094, M1-N1093, M1-A1092, M1-V1091,M1-L1090, M1-L1089, M1-Y1088, M1-C1087, M1-A1086, M1-M1085, M1-I1084,M1-A1083, M1-P1082, M1-V1081, M1-I1080, M1-W1079, M1-A1078, M1-G1077,M1-T1076, M1-K1075, M1-C1074, M1-P1073, M1-P1072, M1-L1071, M1-Q1070,M1-I1069, M1-I1068, M1-K1067, M1-G1066, M1-D1065, M1-E1064, M1-R1063,M1-T1062, M1-E1061, M1-N1060, M1-Q1059, M1-G1058, M1-C1057, M1-P1056,M1-P1055, M1-D1054, M1-I1053, M1-Q1052, M1-D1051, M1-A1050, M1-F1049,M1-V1048, M1-E1047, M1-G1046, M1-Y1045, M1-I1044, M1-M1043, M1-W1042,M1-Y1041, M1-P1040, M1-M1039, M1-Y1038, M1-F1037, M1-I1036, M1-N1035,M1-K1034, M1-A1033, M1-L1032, M1-K1031, M1-W1030, M1-S1029, M1-P1028,M1-E1027, M1-E1026, M1-N1025, M1-P1024, M1-F1023, M1-L1022, M1-I1021,M1-A1020, M1-Q1019, M1-R1018, M1-A1017, M1-V1016, M1-G1015, M1-F1014,M1-S1013, M1-M1012, M1-L1011, M1-V1010, M1-V1009, M1-L1008, M1-M1007,M1-I1006, M1-I1005, M1-V1004, M1-F1003, M1-Y1002, M1-M1001, M1-M1000,M1-D999, M1-I998, M1-M997, M1-M996, M1-K995, M1-G994, M1-I993, M1-M992,M1-M991, M1-V990, M1-Y989, M1-P988, M1-G987, M1-L986, M1-Y985, M1-K984,M1-N983, M1-V982, M1-G981, M1-F980, M1-I979, M1-D978, M1-L977, M1-L976,M1-R975, M1-I974, M1-Y973, M1-W972, M1-Y971, M1-I970, M1-I969, M1-N968,M1-V967, M1-C966, M1-Y965, M1-I964, M1-V963, M1-R962, M1-G961, M1-D960,M1-S959, M1-R958, M1-F957, M1-P956, M1-Q955, M1-D954, M1-Q953, M1-L952,M1-R951, M1-L950, M1-I949, M1-M948, M1-G947, M1-V946, M1-S945, M1-F944,M1-L943, M1-L942, M1-I941, M1-A940, M1-I939, M1-L938, M1-D937, M1-T936,M1-V935, M1-N934, M1-W933, M1-Y932, M1-E931, M1-Q930, M1-L929, M1-W928,M1-V927, M1-K926, M1-V925, M1-K924, M1-Q923, M1-L922, M1-L921, M1-K920,M1-G919, M1-P918, M1-E917, M1-S916, M1-M915, M1-L914, M1-I913, M1-E912,M1-R911, M1-M910, M1-K909, M1-E908, M1-I907, M1-G906, M1-L905, M1-T904,M1-F903, M1-I902, M1-Y901, M1-S900, M1-I899, M1-V898, M1-I897, M1-W896,M1-E895, M1-Q894, M1-T893, M1-S892, M1-P891, M1-W890, M1-R889, M1-E888,M1-M887, M1-K886, M1-V885, M1-L884, M1-V883, M1-I882, M1-Y881, M1-N880,M1-F879, M1-L878, M1-M877, M1-L876, M1-Y875, M1-G874, M1-I873, M1-Y872,M1-A871, M1-L870, M1-T869, M1-Y868, M1-F867, M1-W866, M1-F865, M1-K864,M1-V863, M1-I862, M1-P861, M1-A860, M1-N859, M1-Y858, M1-F857, M1-E856,M1-Y855, M1-I854, M1-K853, M1-R852, M1-G851, M1-L850, M1-P849, M1-I848,M1-L847, M1-R846, M1-H845, M1-K844, M1-S843, M1-Q842, M1-V841, M1-E840,M1-E839, M1-E838, M1-D837, M1-K836, M1-K835, M1-R834, M1-S833, M1-S832,M1-E831, M1-G830, M1-N829, M1-N828, M1-R827, M1-G826, M1-L825, M1-M824,M1-A823, M1-T822, M1-L821, M1-E820, M1-M819, M1-D818, M1-E817, M1-E816,M1-E815, M1-K814, M1-E813, M1-K812, M1-T811, M1-P810, M1-K809, M1-E808,M1-P807, M1-E806, M1-E805, M1-A804, M1-E803, M1-K802, M1-E801, M1-Q800,M1-L799, M1-H798, M1-I797, M1-E796, M1-Q795, M1-A794, M1-Q793, M1-S792,M1-M791, M1-Y790, M1-P789, M1-M788, M1-D787, M1-D786, M1-K785, M1-N784,M1-K783, M1-F782, M1-E781, M1-L780, M1-S779, M1-L778, M1-I777, M1-S776,M1-P775, M1-P774, M1-L773, M1-L772, M1-I771, M1-G770, M1-L769, M1-I768,M1-V767, M1-K766, M1-L765, M1-G764, M1-S763, M1-N762, M1-K761, M1-R760,M1-M759, M1-R758, M1-L757, M1-R756, M1-G755, M1-M754, M1-W753, M1-M752,M1-D751, M1-T750, M1-L749, M1-L748, M1-M747, M1-Q746, M1-S745, M1-C744,M1-T743, M1-H742, M1-A741, M1-I740, M1-F739, M1-D738, M1-R737, M1-H736,M1-K735, M1-A734, M1-A733, M1-V732, M1-A731, M1-L730, M1-Q729, M1-L728,M1-C727, M1-T726, M1-A725, M1-N724, M1-S723, M1-W722, M1-N721, M1-K720,M1-L719, M1-E718, M1-Y717, M1-T716, M1-L715, M1-L714, M1-K713, M1-M712,M1-A711, M1-L710, M1-Q709, M1-E708, M1-D707, M1-Q706, M1-K705, M1-Y704,M1-S703, M1-Q702, M1-D701, M1-L700, M1-L699, M1-E698, M1-V697, M1-A696,M1-L695, M1-Q694, M1-G693, M1-F692, M1-D691, M1-R690, M1-S689, M1-N688,M1-H687, M1-N686, M1-L685, M1-E684, M1-Q683, M1-S682, M1-I681, M1-D680,M1-D679, M1-V678, M1-M677, M1-D676, M1-N675, M1-E674, M1-S673, M1-A672,M1-E671, M1-H670, M1-A669, M1-M668, M1-A667, M1-K666, M1-C665, M1-L664,M1-K663, M1-C662, M1-A661, M1-V660, M1-L659, M1-A658, M1-K657, M1-A656,M1-M655, M1-A654, M1-E653, M1-E652, M1-G651, M1-H650, M1-Q649, M1-W648,M1-F647, M1-F646, M1-L645, M1-A644, M1-M643, M1-K642, M1-Q641, M1-R640,M1-K639, M1-M638, M1-L637, M1-V636, M1-A635, M1-W634, M1-V633, M1-M632,M1-L631, M1-E630, M1-H629, M1-F628, M1-P627, M1-F626, M1-P625, M1-F624,M1-H623, M1-N622, M1-I621, M1-E620, M1-P619, M1-D618, M1-D617, M1-L616,M1-D615, M1-I614, M1-D613, M1-V612, M1-E611, M1-E610, M1-E609, M1-R608,M1-K607, M1-K606, M1-T605, M1-T604, M1-K603, M1-R602, M1-G601, M1-R600,M1-R599, M1-L598, M1-P597, M1-I596, M1-D595, M1-D594, M1-R593, M1-K592,M1-P591, M1-G590, M1-F589, M1-L588, M1-N587, M1-H586, M1-Y585, M1-L584,M1-T583, M1-R582, M1-F581, M1-R580, M1-K579, M1-R578, M1-T577, M1-Y576,M1-N575, M1-C574, M1-R573, M1-Y572, M1-A571, M1-G570, M1-G569, M1-M568,M1-L567, M1-Y566, M1-E565, M1-I564, M1-V563, M1-L562, M1-G561, M1-I560,M1-D559, M1-I558, M1-L557, M1-S556, M1-I555, M1-R554, M1-Y553, M1-D552,M1-P551, M1-P550, M1-L549, M1-N548, M1-G547, M1-K546, M1-K545, M1-V544,M1-D543, M1-R542, M1-V541, M1-L540, M1-H539, M1-Y538, M1-L537, M1-T536,M1-N535, M1-S534, M1-P533, M1-G532, M1-H531, M1-R530, M1-T529, M1-N528,M1-Y527, M1-L526, M1-E525, M1-E524, M1-L523, M1-R522, M1-S521, M1-I520,M1-T519, M1-L518, M1-F517, M1-R516, M1-H515, M1-M514, M1-S513, M1-V512,M1-G511, M1-N510, M1-E509, M1-I508, M1-L507, M1-L506, M1-K505, M1-V504,M1-F503, M1-D502, M1-V501, M1-R500, M1-D499, M1-L498, M1-V497, M1-L496,M1-A495, M1-D494, M1-L493, M1-M492, M1-A491, M1-Q490, M1-E489, M1-L488,M1-S487, M1-G486, M1-V485, M1-P484, M1-W483, M1-Q482, M1-Q481, M1-G480,M1-Y479, M1-I478, M1-F477, M1-I476, M1-Q475, M1-S474, M1-R473, M1-A472,M1-I471, M1-D470, M1-V469, M1-R468, M1-N467, M1-W466, M1-A465, M1-L464,M1-A463, M1-L462, M1-S461, M1-L460, M1-Q459, M1-D458, M1-P457, M1-A456,M1-S455, M1-A454, M1-N453, M1-A452, M1-G451, M1-K450, M1-L449, M1-L448,M1-A447, M1-T446, M1-L445, M1-I444, M1-A443, M1-L442, M1-D441, M1-I440,M1-D439, M1-Q438, M1-H437, M1-G436, M1-E435, M1-S434, M1-G433, M1-M432,M1-R431, M1-F430, M1-V429, M1-T428, M1-I427, M1-L426, M1-E425, M1-K424,M1-K423, M1-K422, M1-M421, M1-C420, M1-E419, M1-M418, M1-L417, M1-I416,M1-I415, M1-F414, M1-L413, M1-H412, M1-Q411, M1-A410, M1-Q409, M1-T408,M1-R407, M1-T406, M1-Y405, M1-T404, M1-F403, M1-T402, M1-K401, M1-Q400,M1-I399, M1-T398, M1-V397, M1-L396, M1-L395, M1-Q394, M1-D393, M1-R392,M1-L391, M1-S390, M1-E389, M1-N388, M1-I387, M1-L386, M1-G385, M1-G384,M1-E383, M1-E382, M1-S381, M1-Y380, M1-K379, M1-H378, M1-G377, M1-F376,M1-A375, M1-L374, M1-I373, M1-D372, M1-S371, M1-A370, M1-R369, M1-G368,M1-S367, M1-G366, M1-D365, M1-C364, M1-V363, M1-V362, M1-V361, M1-P360,M1-V359, M1-P358, M1-P357, M1-T356, M1-D355, M1-R354, M1-L353, M1-Y352,M1-E351, M1-L350, M1-V349, M1-I348, M1-S347, M1-I346, M1-V345, M1-N344,M1-P343, M1-G342, M1-G341, M1-E340, M1-V339, M1-I338, M1-L337, M1-A336,M1-V335, M1-V334, M1-P333, M1-V332, M1-G331, M1-Q330, M1-G329, M1-I328,M1-R327, M1-T326, M1-N325, M1-I324, M1-K323, M1-Q322, M1-L321, M1-S320,M1-I319, M1-H318, M1-K317, M1-E316, M1-L315, M1-Q314, M1-R313, M1-R312,M1-L311, M1-K310, M1-V309, M1-E308, M1-A307, M1-G306, M1-Y305, M1-K304,M1-G303, M1-T302, M1-T301, M1-G300, M1-N299, M1-D298, M1-A297, M1-L296,M1-I295, M1-F294, M1-H293, M1-S292, M1-H291, M1-M290, M1-S289, M1-N288,M1-L287, M1-V286, M1-T285, M1-L284, M1-K283, M1-S282, M1-M281, M1-P280,M1-N279, M1-S278, M1-M277, M1-T276, M1-Q275, M1-Y274, M1-P273, M1-R272,M1-V271, M1-V270, M1-D269, M1-R268, M1-G267, M1-I266, M1-L265, M1-D264,M1-E263, M1-Q262, M1-N261, M1-E260, M1-V259, M1-I258, M1-G257, M1-W256,M1-P255, M1-A254, M1-I253, M1-G252, M1-I251, M1-T250, M1-C249, M1-I248,M1-K247, M1-G246, M1-R245, M1-S244, M1-K243, M1-S242, M1-A241, M1-H240,M1-D239, M1-K238, M1-L237, M1-A236, M1-D235, M1-G234, M1-V233, M1-H232,M1-R231, M1-I230, M1-V229, M1-G228, M1-T227, M1-N226, M1-V225, M1-G224,M1-G223, M1-T222, M1-F221, M1-I220, M1-W219, M1-A218, M1-G217, M1-T216,M1-T215, M1-M214, M1-A213, M1-A212, M1-K211, M1-I210, M1-L209, M1-G208,M1-K207, M1-G206, M1-F205, M1-V204, M1-Q203, M1-K202, M1-L201, M1-K200,M1-P199, M1-Q198, M1-L197, M1-E196, M1-F195, M1-N194, M1-Q193, M1-L192,M1-G191, M1-G190, M1-H189, M1-V188, M1-S187, M1-I186, M1-L185, M1-L184,M1-K183, M1-P182, M1-L181, M1-E180, M1-L179, M1-Q178, M1-W177, M1-E176,M1-K175, M1-T174, M1-M173, M1-L172, M1-H171, M1-L170, M1-L169, M1-L168,M1-D167, M1-P166, M1-K165, M1-T164, M1-D163, M1-F162, M1-S161, M1-V160,M1-R159, M1-V158, M1-Y157, M1-M156, M1-A155, M1-K154, M1-N153, M1-S152,M1-H151, M1-G150, M1-G149, M1-G148, M1-Q147, M1-F146, M1-E145, M1-I144,M1-T143, M1-G142, M1-F141, M1-A140, M1-D139, M1-T138, M1-P137, M1-S136,M1-L135, M1-Q134, M1-T133, M1-H132, M1-K131, M1-S130, M1-I129, M1-S128,M1-W127, M1-K126, M1-E125, M1-S124, M1-Q123, M1-I122, M1-D121, M1-N120,M1-R119, M1-S118, M1-L117, M1-R116, M1-S115, M1-E114, M1-N113, M1-K112,M1-E111, M1-N110, M1-Q109, M1-L108, M1-V107, M1-S106, M1-I105, M1-S104,M1-P103, M1-T102, M1-L101, M1-G100, M1-V99, M1-H98, M1-Q97, M1-G96,M1-I95, M1-L94, M1-R93, M1-G92, M1-C91, M1-C90, M1-C89, M1-R88, M1-H87,M1-P86, M1-D85, M1-K84, M1-T83, M1-S82, M1-P81, M1-I80, M1-I79, M1-H78,M1-V77, M1-C76, M1-E75, M1-R74, M1-K73, M1-Y72, M1-F71, M1-A70, M1-R69,M1-E68, M1-I67, M1-W66, M1-S65, M1-K64, M1-Q63, M1-A62, M1-P61, M1-T60,M1-Q59, M1-Q58, M1-S57, M1-P56, M1-L55, M1-G54, M1-S53, M1-R52, M1-K51,M1-T50, M1-S49, M1-G48, M1-Q47, M1-K46, M1-S45, M1-S44, M1-L43, M1-R42,M1-K41, M1-W40, M1-S39, M1-E38, M1-A37, M1-F36, M1-R35, M1-G34, M1-R33,M1-S32, M1-A31, M1-S30, M1-R29, M1-S28, M1-R27, M1-R26, M1-R25, M1-S24,M1-E23, M1-A22, M1-N21, M1-D20, M1-E19, M1-R18, M1-D17, M1-S16, M1-C15,M1-G14, M1-R13, M1-E12, M1-M11, M1-E10, M1-A9, M1-A8, and/or M1-D7 ofSEQ ID NO:8. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal LTRPC3j deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0449] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3j polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3j polypeptide deletions) of SEQ ID NO:8.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3j (SEQ ID NO:8), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3j (SEQ ID NO:8).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

[0450] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:7 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5157 ofSEQ ID NO:7, b is an integer between 15 to 5171, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:7,and where b is greater than or equal to a+14.

[0451] Features of the Polypeptide Encoded by Gene No:5

[0452] The polypeptide of this gene provided as SEQ ID NO:10 (FIGS.5A-F), encoded by the polynucleotide sequence according to SEQ ID NO:9(FIGS. 5A-F), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3k, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3kpolypeptide with this protein is provided in FIGS. 7A-G.

[0453] The LTRPC3k polypeptide was determined to share 65.5% identityand 73.3% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0454] The LTRPC3k protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0455] Specifically, the LTRPC3k (SEQ ID NO:10) polypeptide represents anovel variant of the LTRPC3e (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:20; and also co-pending U.S. Ser. No. 10/405,793,filed Mar. 28, 2003; which are hereby incorporated by reference hereinin their entirety) polypeptide. The LTRPC3k represents a novel member ofthe TRPM subfamily.

[0456] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0457] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art, particularly LTRPC3e, and LTRPC3.

[0458] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3k polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 5A-F. The transmembrane domains arelocated from about amino acid 777 to about amino acid 794 (TM1), fromabout amino acid 874 to about amino acid 891 (TM2), from about aminoacid 947 to about amino acid 960 (TM3), from about amino acid 974 toabout amino acid 991 (TM4), from about amino acid 1008 to about aminoacid 1025 (TM5), and/or from about amino acid 1095 to about amino acid1115 (TM6) of SEQ ID NO:10. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0459] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:78), IVKFWFYTLAYIGYLMLF (SEQ ID NO:79),VTDLIAILLFSVGM (SEQ ID NO:80), RVIYCVNIIYWYIRLLDI (SEQ ID NO:81),MMIDMMYFVIIMLVVLMS (SEQ ID NO:82), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:83). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3ktransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0460] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3k, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3k polypeptide,particularly for the development of antibodies specific to theseregions.

[0461] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:84),NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:85),ILRLQDQPFRSDG (SEQ ID NO:86), FGVNKYLGPYVMMIGK (SEQ ID NO:87), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDRKQVYDSHTPKSAPCGQNETREDGKIIQLPPCKTGAWIVP (SEQ ID NO:88). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of the LTRPC3k inter-transmembrane polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0462] The LTRPC3k polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 364, 420, 586, 674, 739, 756,978, 1069, 1086, 1099, 1202, 1293, and 1559 of SEQ ID NO:10 (FIGS.5A-F). Conservation of cysteines at key amino acid residues isindicative of conserved structural features, which may correlate withconservation of protein function and/or activity.

[0463] In confirmation of the LTRPC3k representing a member of thetransient receptor channel family, the LTRPC3k polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1130 to about amino acid 1135 of SEQ ID NO:10. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0464] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:89).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3k TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0465] In further confirmation of the LTRPC3k representing a member ofthe transient receptor channel family, the LTRPC3k polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 905 to about amino acid 1116 of SEQ IDNO:10. In this context, the term “about” may be construed to mean 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/orC-terminus of the above referenced polypeptide.

[0466] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:90).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3k ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0467] The LTRPC3k polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1220 to aboutamino acid 1275 of SEQ ID NO:10. In this context, the term “about” maybe construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0468] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:91).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3k coiled-coil domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0469] LTRPC3k polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3k by identifying mutations in the LTRPC3k gene using LTRPC3ksequences as probes or by determining LTRPC3k protein or mRNA expressionlevels. LTRPC3k polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3k peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3k.

[0470] Since the LTRPC3k polypeptide represents an N-terminally extendedvariant of the LTRPC3e polypeptide (see polypeptide alignment providedin FIG. 11A-I), and since the LTRPC3e (SEQ ID NO:20) polypeptiderepesents a splice variant of the LTRPC3 polypeptide (SEQ ID NO:15), theLTRPC3k (SEQ ID NO:10) polypeptide of the present invention is expectedto have the same or similar function as the LTRPC3 polypeptide, and inparticular, the LTRPC3e polypeptide. Specifically, the LTRPC3kpolypeptide is expected to localize to the cell membrane.

[0471] Moreover, the LTRPC3k polypeptide is expected to also localize inor near the plasmalemmal compartment.

[0472] Moreover, the LTRPC3k polypeptide is also expected to be aconstitutively active channel capable of mediating Ca²⁺ influx.

[0473] Moreover, the LTRPC3k polypeptide is also expected to mediateCa²⁺ entry and that LTRPC3k-mediated Ca²⁺ entry is expected to bepotentiated by store-depletion.

[0474] Moreover, the LTRPC3k polypeptide is also expected to mediate aCa²⁺ entry pathway that is distinct from the endogenous Ca²⁺ entrypathways present in HEK 293 cells.

[0475] Moreover, the LTRPC3k polypeptide of the present invention isexpected to share the same or similar expression profile as that of theLTRPC3 polypeptide (SEQ ID NO:15).

[0476] Moreover, the LTRPC3k polypeptide is expected to share the sameor similar differential expression patterns observed for the LTRPC3variant.

[0477] Moreover, the LTRCP3g polypeptide is expected to be able tomodulate the FEN1 DNA base-excision repair/proliferation modulatingprotein to the same or similar extent as the LTRPC3 polypeptide.

[0478] Moreover, the LTRPC3k polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein, such as LTRPC3 (SEQ ID NO:15), andparticularly as LTRPC3b (SEQ ID NO:17).

[0479] Moreover, the LTRPC3k polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein, such as the LTRPC3 variant (SEQ IDNO:15), and particularly as LTRPC3e (SEQ ID NO:20).

[0480] In preferred embodiments, LTRPC3k polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0481] In preferred embodiments, LTRPC3k polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0482] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0483] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0484] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3k to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3k activity or a compound that increased LTRPC3k message levelswould be a desired invention for cancer therapy.

[0485] In preferred embodiments, LTRPC3k polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0486] Moreover, LTRPC3k polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0487] In preferred embodiments, antagonists directed against LTRPC3kare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0488] Moreover, antagonists directed against LTRPC3k are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0489] In preferred embodiments, agonists directed against LTRPC3k areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0490] Moreover, agonists directed against LTRPC3k are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0491] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3kpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0492] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0493] Thus, the LTRPC3k polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0494] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3kpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0495] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3kpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0496] In preferred embodiments, LTRPC3k polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3kpolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0497] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3k polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0498] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0499] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0500] Thus, the LTRPC3k polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0501] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0502] In preferred embodiments, LTRPC3k polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0503] In more preferred embodiments, LTRPC3k polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0504] LTRPC3k polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3kpolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0505] The LTRPC3k gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3k can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0506] Moreover, given the selective expression in kidney, LTRPC3k couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3k chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3k is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3k inthe disease. Therefore, it is possible that LTRPC3k may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0507] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3k may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3k may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0508] In addition, LTRPC3k polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0509] The LTRPC3k polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3k polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0510] In addition, antagonists of the LTRPC3k polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0511] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3k polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3k, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0512] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3k gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0513] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3k, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3k and assessing their ability to grow wouldprovide convincing evidence the LTRPC3k polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0514] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0515] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0516] In the case of LTRPC3k transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0517] In preferred embodiments, the following N-terminal LTRPC3kdeletion polypeptides are encompassed by the present invention:M1-T1711, G2-T1711, K3-T1711, K4-T1711, W5-T1711, R6-T1711, D7-T1711,A8-T1711, A9-T1711, E10-T1711, M11-T1711, E12-T1711, R13-T1711,G14-T1711, C15-T1711, S16-T1711, D17-T1711, R18-T1711, E19-T1711,D20-T1711, N21-T1711, A22-T1711, E23-T1711, S24-T1711, R25-T1711,R26-T1711, R27-T1711, S28-T1711, R29-T1711, S30-T1711, A31-T1711,S32-T1711, R33-T1711, G34-T1711, R35-T1711, F36-T1711, A37-T1711,E38-T1711, S39-T1711, W40-T1711, K41-T1711, R42-T1711, L43-T1711,S44-T1711, S45-T1711, K46-T1711, Q47-T1711, G48-T1711, S49-T1711,T50-T1711, K51-T1711, R52-T1711, S53-T1711, G54-T1711, L55-T1711,P56-T1711, S57-T1711, Q58-T1711, Q59-T1711, T60-T1711, P61-T1711,A62-T1711, Q63-T1711, K64-T1711, S65-T1711, W66-T1711, I67-T1711,E68-T1711, R69-T1711, A70-T1711, F71-T1711, Y72-T1711, K73-T1711,R74-T1711, E75-T1711, C76-T1711, V77-T1711, H78-T1711, I79-T1711,I80-T1711, P81-T1711, S82-T1711, T83-T1711, K84-T1711, D85-T1711,P86-T1711, H87-T1711, R88-T1711, C89-T1711, C90-T1711, C91-T1711,G92-T1711, R93-T1711, L94-T1711, I95-T1711, G96-T1711, Q97-T1711,H98-T1711, V99-T1711, G100-T1711, L10-T1711, T102-T1711, P103-T1711,S104-T1711, I105-T1711, S106-T1711, V107-T1711, L108-T1711, Q109-T1711,N110-T1711, E111-T1711, K112-T1711, N113-T1711, E114-T1711, S115-T1711,R116-T1711, L117-T1711, S118-T1711, R119-T1711, N120-T1711, D121-T1711,I122-T1711, Q123-T1711, S124-T1711, E125-T1711, K126-T1711, W127-T1711,S128-T1711, I129-T1711, S130-T1711, K131-T1711, H132-T1711, T133-T1711,Q134-T1711, L135-T1711, S136-T1711, P137-T1711, T138-T1711, D139-T1711,A140-T1711, F141-T1711, G142-T1711, T143-T1711, I144-T1711, E145-T1711,F146-T1711, Q147-T1711, G148-T1711, G149-T1711, G150-T1711, H151-T1711,S152-T1711, N153-T1711, K154-T1711, and/or A155-T1711 of SEQ ID NO:10.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3k deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0518] In preferred embodiments, the following C-terminal LTRPC3kdeletion polypeptides are encompassed by the present invention:M1-T1711, M1-H1710, M1-K1709, M1-S1708, M1-E1707, M1-F1706, M1-S1705,M1-Q1704, M1-F1703, M1-A1702, M1-S1701, M1-T1700, M1-R1699, M1-S1698,M1-L1697, M1-R1696, M1-R1695, M1-M1694, M1-S1693, M1-L1692, M1-S1691,M1-D1690, M1-G1689, M1-R1688, M1-G1687, M1-E1686, M1-P1685, M1-K1684,M1-S1683, M1-S1682, M1-K1681, M1-S1680, M1-R1679, M1-Q1678, M1-F1677,M1-P1676, M1-N1675, M1-Q1674, M1-L1673, M1-S1672, M1-A1671, M1-T1670,M1-N1669, M1-R1668, M1-Q1667, M1-R1666, M1-D1665, M1-L1664, M1-K1663,M1-D1662, M1-S1661, M1-I1660, M1-S1659, M1-F1658, M1-S1657, M1-K1656,M1-R1655, M1-T1654, M1-H1653, M1-A1652, M1-Y1651, M1-P1650, M1-A1649,M1-S1648, M1-P1647, M1-E1646, M1-E1645, M1-A1644, M1-S1643, M1-Y1642,M1-S1641, M1-N1640, M1-A1639, M1-R1638, M1-E1637, M1-I1636, M1-K1635,M1-P1634, M1-V1633, M1-T1632, M1-I1631, M1-N1630, M1-N1629, M1-S1628,M1-L1627, M1-T1626, M1-R1625, M1-E1624, M1-S1623, M1-N1622, M1-D1621,M1-G1620, M1-E1619, M1-Q1618, M1-S1617, M1-S1616, M1-I1615, M1-A1614,M1-I1613, M1-T1612, M1-A1611, M1-R1610, M1-R1609, M1-G1608, M1-K1607,M1-A1606, M1-E1605, M1-N1604, M1-E1603, M1-E1602, M1-S1601, M1-D1600,M1-S1599, M1-S1598, M1-P1597, M1-H1596, M1-S1595, M1-L1594, M1-E1593,M1-A1592, M1-E1591, M1-R1590, M1-E1589, M1-P1588, M1-H1587, M1-C1586,M1-C1585, M1-T1584, M1-L1583, M1-D1582, M1-E1581, M1-V1580, M1-K1579,M1-D1578, M1-G1577, M1-L1576, M1-G1575, M1-G1574, M1-P1573, M1-F1572,M1-A1571, M1-A1570, M1-R1569, M1-D1568, M1-A1567, M1-I1566, M1-A1565,M1-Q1564, M1-P1563, M1-A1562, M1-N1561, M1-V1560, M1-C1559, M1-R1558,M1-T1557, M1-D1556, M1-I1555, M1-C1554, M1-D1553, M1-T1552, M1-I1551,M1-S1550, M1-T1549, M1-Y1548, M1-E1547, M1-A1546, M1-T1545, M1-K1544,M1-V1543, M1-P1542, M1-V1541, M1-G1540, M1-F1539, M1-N1538, M1-A1537,M1-Y1536, M1-Y1535, M1-S1534, M1-R1533, M1-S1532, M1-P1531, M1-S1530,M1-F1529, M1-M1528, M1-F1527, M1-S1526, M1-H1525, M1-S1524, M1-K1523,M1-V1522, M1-I1521, M1-P1520, M1-A1519, M1-E1518, M1-E1517, M1-L1516,M1-L1515, M1-F1514, M1-P1513, M1-T1512, M1-T1511, M1-A1510, M1-L1509,M1-Y1508, M1-R1507, M1-S1506, M1-S1505, M1-K1504, M1-S1503, M1-R1502,M1-E1501, M1-I1500, M1-T1499, M1-H1498, M1-Y1497, M1-M1496, M1-P1495,M1-P1494, M1-E1493, M1-S1492, M1-D1491, M1-W1490, M1-P1489, M1-N1488,M1-Q1487, M1-C1486, M1-E1485, M1-P1484, M1-L1483, M1-H1482, M1-T1481,M1-Y1480, M1-D1479, M1-S1478, M1-S1477, M1-F1476, M1-S1475, M1-R1474,M1-T1473, M1-D1472, M1-M1471, M1-S1470, M1-T1469, M1-I1468, M1-D1467,M1-E1466, M1-F1465, M1-D1464, M1-I1463, M1-S1462, M1-R1461, M1-S1460,M1-P1459, M1-P1458, M1-R1457, M1-D1456, M1-T1455, M1-P1454, M1-A1453,M1-L1452, M1-T1451, M1-A1450, M1-Y1449, M1-A1448, M1-S1447, M1-S1446,M1-S1445, M1-P1444, M1-A1443, M1-T1442, M1-S1441, M1-P1440, M1-V1439,M1-P1438, M1-T1437, M1-S1436, M1-F1435, M1-S1434, M1-P1433, M1-E1432,M1-G1431, M1-L1430, M1-G1429, M1-L1428, M1-I1427, M1-N1426, M1-V1425,M1-S1424, M1-N1423, M1-D1422, M1-L1421, M1-P1420, M1-D1419, M1-I1418,M1-D1417, M1-C1416, M1-H1415, M1-L1414, M1-E1413, M1-D1412, M1-M1411,M1-A1410, M1-S1409, M1-V1408, M1-Y1407, M1-I1406, M1-D1405, M1-I1404,M1-C1403, M1-S1402, M1-S1401, M1-P1400, M1-R1399, M1-R1398, M1-S1397,M1-D1396, M1-P1395, M1-V1394, M1-I1393, M1-A1392, M1-L1391, M1-T1390,M1-N1389, M1-A1388, M1-P1387, M1-A1386, M1-A1385, M1-P1384, M1-A1383,M1-K1382, M1-P1381, M1-E1380, M1-K1379, M1-A1378, M1-V1377, M1-S1376,M1-H1375, M1-S1374, M1-S1373, M1-T1372, M1-A1371, M1-R1370, M1-H1369,M1-L1368, M1-S1367, M1-L1366, M1-S1365, M1-R1364, M1-E1363, M1-K1362,M1-F1361, M1-I1360, M1-S1359, M1-E1358, M1-L1357, M1-K1356, M1-E1355,M1-I1354, M1-G1353, M1-G1352, M1-K1351, M1-D1350, M1-K1349, M1-M1348,M1-N1347, M1-V1346, M1-S1345, M1-Y1344, M1-F1343, M1-S1342, M1-H1341,M1-S1340, M1-R1339, M1-M1338, M1-R1337, M1-P1336, M1-M1335, M1-L1334,M1-T1333, M1-P1332, M1-S1331, M1-T1330, M1-P1329, M1-S1328, M1-M1327,M1-T1326, M1-E1325, M1-E1324, M1-G1323, M1-A1322, M1-P1321, M1-D1320,M1-I1319, M1-S1318, M1-E1317, M1-Q1316, M1-L1315, M1-K1314, M1-F1313,M1-T1312, M1-N1311, M1-G1310, M1-E1309, M1-Q1308, M1-S1307, M1-N1306,M1-F1305, M1-S1304, M1-S1303, M1-Q1302, M1-R1301, M1-V1300, M1-I1299,M1-Y1298, M1-A1297, M1-A1296, M1-D1295, M1-T1294, M1-C1293, M1-D1292,M1-S1291, M1-S1290, M1-T1289, M1-R1288, M1-S1287, M1-R1286, M1-I1285,M1-K1284, M1-N1283, M1-S1282, M1-E1281, M1-A1280, M1-R1279, M1-E1278,M1-L1277, M1-G1276, M1-T1275, M1-L1274, M1-R1273, M1-E1272, M1-L1271,M1-A1270, M1-T1269, M1-A1268, M1-M1267, M1-R1266, M1-G1265, M1-I1264,M1-L1263, M1-D1262, M1-E1261, M1-L1260, M1-Q1259, M1-A1258, M1-L1257,M1-R1256, M1-I1255, M1-D1254, M1-V1253, M1-T1252, M1-Q1251, M1-L1250,M1-S1249, M1-A1248, M1-K1247, M1-M1246, M1-S1245, M1-H1244, M1-E1243,M1-R1242, M1-E1241, M1-N1240, M1-V1239, M1-E1238, M1-E1237, M1-L1236,M1-R1235, M1-M1234, M1-S1233, M1-M1232, M1-N1231, M1-E1230, M1-V1229,M1-R1228, M1-E1227, M1-S1226, M1-T1225, M1-V1224, M1-R1223, M1-I1222,M1-R1221, M1-E1220, M1-D1219, M1-N1218, M1-S1217, M1-S1216, M1-N1215,M1-F1214, M1-R1213, M1-D1212, M1-D1211, M1-K1210, M1-E1209, M1-R1208,M1-F1207, M1-Y1206, M1-E1205, M1-E1204, M1-I1203, M1-C1202, M1-Q1201,M1-E1200, M1-E1199, M1-F1198, M1-D1197, M1-H1196, M1-V1195, M1-K1194,M1-K1193, M1-L1192, M1-E1191, M1-D1190, M1-D1189, M1-T1188, M1-I1187,M1-F1186, M1-L1185, M1-K1184, M1-L1183, M1-G1182, M1-Y1181, M1-D1180,M1-R1179, M1-E1178, M1-D1177, M1-P1176, M1-D1175, M1-S1174, M1-E1173,M1-H1172, M1-K1171, M1-R1170, M1-W1169, M1-R1168, M1-C1167, M1-C1166,M1-L1165, M1-H1164, M1-Q1163, M1-F1162, M1-I1161, M1-M1160, M1-T1159,M1-M1158, M1-H1157, M1-S1156, M1-F1155, M1-I1154, M1-I1153, M1-L1152,M1-P1151, M1-P1150, M1-P1149, M1-L1148, M1-V1147, M1-P1146, M1-R1145,M1-E1144, M1-H1143, M1-F1142, M1-T1141, M1-M1140, M1-I1139, M1-L1138,M1-Q1137, M1-Y1136, M1-R1135, M1-Q1134, M1-F1133, M1-K1132, M1-W 131,M1-V1130, M1-Q1129, M1-N1128, M1-S1127, M1-I1126, M1-S1125, M1-K1124,M1-V1123, M1-E1122, M1-F1121, M1-F1120, M1-T1119, M1-N1118, M1-N1117,M1-F1116, M1-V1115, M1-A1114, M1-I1113, M1-L1112, M1-L1111, M1-N1110,M1-V1109, M1-L1108, M1-L1107, M1-I1106, M1-N1105, M1-A1104, M1-V1103,M1-L1102, M1-L1101, M1-Y1100, M1-C1099, M1-A1098, M1-M1097, M1-I1096,M1-A1095, M1-P1094, M1-V1093, M1-I1092, M1-W1091, M1-A1090, M1-G1089,M1-T1088, M1-K1087, M1-C1086, M1-P1085, M1-P1084, M1-L1083, M1-Q1082,M1-I1081, M1-I1080, M1-K1079, M1-G1078, M1-D1077, M1-E1076, M1-R1075,M1-T1074, M1-E1073, M1-N1072, M1-Q1071, M1-G1070, M1-C1069, M1-P1068,M1-P1067, M1-D1066, M1-I1065, M1-Q1064, M1-D1063, M1-A1062, M1-F1061,M1-V1060, M1-E1059, M1-G1058, M1-Y1057, M1-I1056, M1-M1055, M1-W1054,M1-Y1053, M1-P1052, M1-M1051, M1-Y1050, M1-F1049, M1-I1048, M1-N1047,M1-K1046, M1-A1045, M1-L1044, M1-K1043, M1-W1042, M1-S1041, M1-P1040,M1-E1039, M1-E1038, M1-N1037, M1-P1036, M1-F1035, M1-L1034, M1-I1033,M1-A1032, M1-Q1031, M1-R1030, M1-A1029, M1-V1028, M1-G1027, M1-F1026,M1-S1025, M1-M1024, M1-L1023, M1-V1022, M1-V1021, M1-L1020, M1-M1019,M1-I1018, M1-I1017, M1-V1016, M1-F1015, M1-Y1014, M1-M1013, M1-M1012,M1-D1001, M1-I1010, M1-M1009, M1-M1008, M1-K1007, M1-G1006, M1-I1005,M1-M1004, M1-M1003, M1-V1002, M1-Y1001, M1-P1000, M1-G999, M1-L998,M1-Y997, M1-K996, M1-N995, M1-V994, M1-G993, M1-F992, M1-I991, M1-D990,M1-L989, M1-L988, M1-R987, M1-I986, M1-Y985, M1-W984, M1-Y983, M1-I982,M1-I981, M1-N980, M1-V979, M1-C978, M1-Y977, M1-I976, M1-V975, M1-R974,M1-G973, M1-D972, M1-S971, M1-R970, M1-F969, M1-P968, M1-Q967, M1-D966,M1-Q965, M1-L964, M1-R963, M1-L962, M1-I961, M1-M960, M1-G959, M1-V958,M1-S957, M1-F956, M1-L955, M1-L954, M1-I953, M1-A952, M1-I951, M1-L950,M1-D949, M1-T948, M1-V947, M1-N946, M1-W945, M1-Y944, M1-E943, M1-Q942,M1-L941, M1-W940, M1-V939, M1-K938, M1-V937, M1-K936, M1-Q935, M1-L934,M1-L933, M1-K932, M1-G931, M1-P930, M1-E929, M1-S928, M1-M927, M1-L926,M1-I925, M1-E924, M1-R923, M1-M922, M1-K921, M1-E920, M1-I919, M1-G918,M1-L917, M1-T916, M1-F915, M1-I914, M1-Y913, M1-S912, M1-I911, M1-V910,M1-I909, M1-W908, M1-E907, M1-Q906, M1-T905, M1-S904, M1-P903, M1-W902,M1-R901, M1-E900, M1-M899, M1-K898, M1-V897, M1-L896, M1-V895, M1-I894,M1-Y893, M1-N892, M1-F891, M1-L890, M1-M889, M1-L888, M1-Y887, M1-G886,M1-I885, M1-Y884, M1-A883, M1-L882, M1-T881, M1-Y880, M1-F879, M1-W878,M1-F877, M1-K876, M1-V875, M1-I874, M1-P873, M1-A872, M1-N871, M1-Y870,M1-F869, M1-E868, M1-Y867, M1-I866, M1-K865, M1-R864, M1-G863, M1-L862,M1-P861, M1-I860, M1-L859, M1-R858, M1-H857, M1-K856, M1-S855, M1-Q854,M1-V853, M1-E852, M1-E851, M1-E850, M1-D849, M1-K848, M1-K847, M1-R846,M1-S845, M1-S844, M1-E843, M1-G842, M1-N841, M1-N840, M1-R839, M1-G838,M1-L837, M1-M836, M1-A835, M1-T834, M1-L833, M1-E832, M1-M831, M1-D830,M1-E829, M1-E828, M1-E827, M1-K826, M1-E825, M1-K824, M1-T823, M1-P822,M1-K821, M1-E820, M1-P819, M1-E818, M1-E817, M1-A816, M1-E815, M1-K814,M1-E813, M1-Q812, M1-L811, M1-H810, M1-I809, M1-E808, M1-Q807, M1-A806,M1-Q805, M1-S804, M1-M803, M1-Y802, M1-P801, M1-M800, M1-D799, M1-D798,M1-K797, M1-N796, M1-K795, M1-F794, M1-E793, M1-L792, M1-S791, M1-L790,M1-I789, M1-S788, M1-P787, M1-P786, M1-L785, M1-L784, M1-I783, M1-G782,M1-L781, M1-I780, M1-V779, M1-K778, M1-L777, M1-G776, M1-S775, M1-N774,M1-K773, M1-R772, M1-M771, M1-R770, M1-L769, M1-R768, M1-G767, M1-M766,M1-W765, M1-M764, M1-D763, M1-T762, M1-L761, M1-L760, M1-M759, M1-Q758,M1-S757, M1-C756, M1-T755, M1-H754, M1-A753, M1-I752, M1-F751, M1-D750,M1-R749, M1-H748, M1-K747, M1-A746, M1-A745, M1-V744, M1-A743, M1-L742,M1-Q741, M1-L740, M1-C739, M1-T738, M1-A737, M1-N736, M1-S735, M1-W734,M1-N733, M1-K732, M1-L731, M1-E730, M1-Y729, M1-T728, M1-L727, M1-L726,M1-K725, M1-M724, M1-A723, M1-L722, M1-Q721, M1-E720, M1-D719, M1-Q718,M1-K717, M1-Y716, M1-S715, M1-Q714, M1-D713, M1-L712, M1-L711, M1-E710,M1-V709, M1-A708, M1-L707, M1-Q706, M1-G705, M1-F704, M1-D703, M1-R702,M1-S701, M1-N700, M1-H699, M1-N698, M1-L697, M1-E696, M1-Q695, M1-S694,M1-I693, M1-D692, M1-D691, M1-V690, M1-M689, M1-D688, M1-N687, M1-E686,M1-S685, M1-A684, M1-E683, M1-H682, M1-A681, M1-M680, M1-A679, M1-K678,M1-C677, M1-L676, M1-K675, M1-C674, M1-A673, M1-V672, M1-L671, M1-A670,M1-K669, M1-A668, M1-M667, M1-A666, M1-E665, M1-E664, M1-G663, M1-H662,M1-Q661, M1-W660, M1-F659, M1-F658, M1-L657, M1-A656, M1-M655, M1-K654,M1-Q653, M1-R652, M1-K651, M1-M650, M1-L649, M1-V648, M1-A647, M1-W646,M1-V645, M1-M644, M1-L643, M1-E642, M1-H641, M1-F640, M1-P639, M1-F638,M1-P637, M1-F636, M1-H635, M1-N634, M1-I633, M1-E632, M1-P631, M1-D630,M1-D629, M1-L628, M1-D627, M1-I626, M1-D625, M1-V624, M1-E623, M1-E622,M1-E621, M1-R620, M1-K619, M1-K618, M1-T617, M1-T616, M1-K615, M1-R614,M1-G613, M1-R612, M1-R611, M1-L610, M1-P609, M1-I608, M1-D607, M1-D606,M1-R605, M1-K604, M1-P603, M1-G602, M1-F601, M1-L600, M1-N599, M1-H598,M1-Y597, M1-L596, M1-T595, M1-R594, M1-F593, M1-R592, M1-K591, M1-R590,M1-T589, M1-Y588, M1-N587, M1-C586, M1-R585, M1-Y584, M1-A583, M1-G582,M1-G581, M1-M580, M1-L579, M1-Y578, M1-E577, M1-I576, M1-V575, M1-L574,M1-G573, M1-I572, M1-D571, M1-I570, M1-L569, M1-S568, M1-I567, M1-R566,M1-Y565, M1-D564, M1-P563, M1-P562, M1-L561, M1-N560, M1-G559, M1-K558,M1-F557, M1-Y556, M1-I555, M1-W554, M1-G553, M1-F552, M1-G551, M1-P550,M1-Y549, M1-E548, M1-R547, M1-K546, M1-K545, M1-V544, M1-D543, M1-R542,M1-V541, M1-L540, M1-H539, M1-Y538, M1-L537, M1-T536, M1-N535, M1-S534,M1-P533, M1-G532, M1-H531, M1-R530, M1-T529, M1-N528, M1-Y527, M1-L526,M1-E525, M1-E524, M1-L523, M1-R522, M1-S521, M1-I520, M1-T519, M1-L518,M1-F517, M1-R516, M1-H515, M1-M514, M1-S513, M1-V512, M1-G511, M1-N510,M1-E509, M1-I508, M1-L507, M1-L506, M1-K505, M1-V504, M1-F503, M1-D502,M1-V501, M1-R500, M1-D499, M1-L498, M1-V497, M1-L496, M1-A495, M1-D494,M1-L493, M1-M492, M1-A491, M1-Q490, M1-E489, M1-L488, M1-S487, M1-G486,M1-V485, M1-P484, M1-W483, M1-Q482, M1-Q481, M1-G480, M1-Y479, M1-I478,M1-F477, M1-I476, M1-Q475, M1-S474, M1-R473, M1-A472, M1-I471, M1-D470,M1-V469, M1-R468, M1-N467, M1-W466, M1-A465, M1-L464, M1-A463, M1-L462,M1-S461, M1-L460, M1-Q459, M1-D458, M1-P457, M1-A456, M1-S455, M1-A454,M1-N453, M1-A452, M1-G451, M1-K450, M1-L449, M1-L448, M1-A447, M1-T446,M1-L445, M1-I444, M1-A443, M1-L442, M1-D441, M1-I440, M1-D439, M1-Q438,M1-H437, M1-G436, M1-E435, M1-S434, M1-G433, M1-M432, M1-R431, M1-F430,M1-V429, M1-T428, M1-I427, M1-L426, M1-E425, M1-K424, M1-K423, M1-K422,M1-M421, M1-C420, M1-E419, M1-M418, M1-L417, M1-I416, M1-I415, M1-F414,M1-L413, M1-H412, M1-Q411, M1-A410, M1-Q409, M1-T408, M1-R407, M1-T406,M1-Y405, M1-T404, M1-F403, M1-T402, M1-K401, M1-Q400, M1-I399, M1-T398,M1-V397, M1-L396, M1-L395, M1-Q394, M1-D393, M1-R392, M1-L391, M1-S390,M1-E389, M1-N388, M1-I387, M1-L386, M1-G385, M1-G384, M1-E383, M1-E382,M1-S381, M1-Y380, M1-K379, M1-H378, M1-G377, M1-F376, M1-A375, M1-L374,M1-I373, M1-D372, M1-S371, M1-A370, M1-R369, M1-G368, M1-S367, M1-G366,M1-D365, M1-C364, M1-V363, M1-V362, M1-V361, M1-P360, M1-V359, M1-P358,M1-P357, M1-T356, M1-D355, M1-R354, M1-L353, M1-Y352, M1-E351, M1-L350,M1-V349, M1-I348, M1-S347, M1-I346, M1-V345, M1-N344, M1-P343, M1-G342,M1-G341, M1-E340, M1-V339, M1-I338, M1-L337, M1-A336, M1-V335, M1-V334,M1-P333, M1-V332, M1-G331, M1-Q330, M1-G329, M1-I328, M1-R327, M1-T326,M1-N325, M1-I324, M1-K323, M1-Q322, M1-L321, M1-S320, M1-I319, M1-H318,M1-K317, M1-E316, M1-L315, M1-Q314, M1-R313, M1-R312, M1-L311, M1-K310,M1-V309, M1-E308, M1-A307, M1-G306, M1-Y305, M1-K304, M1-G303, M1-T302,M1-T301, M1-G300, M1-N299, M1-D298, M1-A297, M1-L296, M1-I295, M1-F294,M1-H293, M1-S292, M1-H291, M1-M290, M1-S289, M1-N288, M1-L287, M1-V286,M1-T285, M1-L284, M1-K283, M1-S282, M1-M281, M1-P280, M1-N279, M1-S278,M1-M277, M1-T276, M1-Q275, M1-Y274, M1-P273, M1-R272, M1-V271, M1-V270,M1-D269, M1-R268, M1-G267, M1-I266, M1-L265, M1-D264, M1-E263, M1-Q262,M1-N261, M1-E260, M1-V259, M1-I258, M1-G257, M1-W256, M1-P255, M1-A254,M1-I253, M1-G252, M1-I251, M1-T250, M1-C249, M1-I248, M1-K247, M1-G246,M1-R245, M1-S244, M1-K243, M1-S242, M1-A241, M1-H240, M1-D239, M1-K238,M1-L237, M1-A236, M1-D235, M1-G234, M1-V233, M1-H232, M1-R231, M1-I230,M1-V229, M1-G228, M1-T227, M1-N226, M1-V225, M1-G224, M1-G223, M1-T222,M1-F221, M1-I220, M1-W219, M1-A218, M1-G217, M1-T216, M1-T215, M1-M214,M1-A213, M1-A212, M1-K211, M1-I210, M1-L209, M1-G208, M1-K207, M1-G206,M1-F205, M1-V204, M1-Q203, M1-K202, M1-L201, M1-K200, M1-P199, M1-Q198,M1-L197, M1-E196, M1-F195, M1-N194, M1-Q193, M1-L192, M1-G191, M1-G190,M1-H189, M1-V188, M1-S187, M1-I186, M1-L185, M1-L184, M1-K183, M1-P182,M1-L181, M1-E180, M1-L179, M1-Q178, M1-W177, M1-E176, M1-K175, M1-T174,M1-M173, M1-L172, M1-H171, M1-L170, M1-L169, M1-L168, M1-D167, M1-P166,M1-K165, M1-T164, M1-D163, M1-F162, M1-S161, M1-V160, M1-R159, M1-V158,M1-Y157, M1-M156, M1-A155, M1-K154, M1-N153, M1-S152, M1-H151, M1-G150,M1-G149, M1-G148, M1-Q147, M1-F146, M1-E145, M1-I144, M1-T143, M1-G142,M1-F141, M1-A140, M1-D139, M1-T138, M1-P137, M1-S136, M1-L135, M1-Q134,M1-T133, M1-H132, M1-K131, M1-S130, M1-I129, M1-S128, M1-W127, M1-K126,M1-E125, M1-S124, M1-Q123, M1-I122, M1-D121, M1-N120, M1-R119, M1-S118,M1-L117, M1-R116, M1-S115, M1-E114, M1-N113, M1-K112, M1-E111, M1-N110,M1-Q109, M1-L108, M1-V107, M1-S106, M1-I105, M1-S104, M1-P103, M1-T102,M1-L101, M1-G100, M1-V99, M1-H98, M1-Q97, M1-G96, M1-I95, M1-L94,M1-R93, M1-G92, M1-C91, M1-C90, M1-C89, M1-R88, M1-H87, M1-P86, M1-D85,M1-K84, M1-T83, M1-S82, M1-P81, M1-I80, M1-I79, M1-H78, M1-V77, M1-C76,M1-E75, M1-R74, M1-K73, M1-Y72, M1-F71, M1-A70, M1-R69, M1-E68, M1-I67,M1-W66, M1-S65, M1-K64, M1-Q63, M1-A62, M1-P61, M1-T60, M1-Q59, M1-Q58,M1-S57, M1-P56, M1-L55, M1-G54, M1-S53, M1-R52, M1-K51, M1-T50, M1-S49,M1-G48, M1-Q47, M1-K46, M1-S45, M1-S44, M1-L43, M1-R42, M1-K41, M1-W40,M1-S39, M1-E38, M1-A37, M1-F36, M1-R35, M1-G34, M1-R33, M1-S32, M1-A31,M1-S30, M1-R29, M1-S28, M1-R27, M1-R26, M1-R25, M1-S24, M1-E23, M1-A22,M1-N21, M1-D20, M1-E19, M1-R18, M1-D17, M1-S16, M1-C15, M1-G14, M1-R13,M1-E12, M1-M11, M1-E10, M1-A9, M1-A8, and/or M1-D7 of SEQ ID NO:10.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these C-terminalLTRPC3k deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0519] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3k polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3k polypeptide deletions) of SEQ ID NO:10.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3k (SEQ ID NO:10), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3k (SEQ IDNO:10). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

[0520] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:9 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5193ofSEQ ID NO:9, b is an integer between 15 to 5207, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:9,and where b is greater than or equal to a+14.

[0521] Features of the Polypeptide Encoded by Gene No:5

[0522] The polypeptide of this gene provided as SEQ ID NO:12 (FIGS.6A-F), encoded by the polynucleotide sequence according to SEQ ID NO:11(FIGS. 6A-F), and/or encoded by the polynucleotide contained within thedeposited clone, LTRPC3l, has significant homology at the nucleotide andamino acid level to the human melastatin 1 protein (Melastatin1; GenbankAccession No. gi|3243075; SEQ ID NO:13). An alignment of the LTRPC3lpolypeptide with this protein is provided in FIGS. 7A-G.

[0523] The LTRPC3l polypeptide was determined to share 65.7% identityand 73.5% similarity with the human melastatin 1 protein (Melastatin1;Genbank Accession No. gi|3243075; SEQ ID NO:13) as shown in FIG. 10.

[0524] The LTRPC3l protein is believed to represent a member of a newclass of protein kinases referred to as alpha kinases (Curr. Biol. 9(2), R43-R45 (1999)). These kinases represent a novel type of signalingmolecule comprising both a catalytic protein kinase domain, in additionto, an ion channel domain. This family is commonly referred to as thetransient receptor potential channel (TRP) family. Melastatin1 defines aseparate subfamily of TRP channels referred to as TRPM (melastatin1).TRPM family members are characteristic of their unusually longcytoplasmic tails at both ends of the channel domain and some of thefamily members contain an enzyme domain at the C-terminal region.

[0525] Specifically, the LTRPC3l (SEQ ID NO:12) polypeptide represents anovel variant of the LTRPC3f (Co-pending U.S. Ser. No. 10/210,152, filedAug. 1, 2002; International Publication No. WO 03/012063, published Feb.13, 2003; SEQ ID NO:21) polypeptide. The LTRPC3l represents a novelmember of the TRPM subfamily.

[0526] The melastatin1 protein is believed to be negatively associatedwith the incidence of melanoma based upon its inverse correlativeexpression in highly aggressive melanomas (Genomics 54 (1), 116-123(1998)). Thus, overexpression of melastatin1 could represent a noveltherapeutic in the treatment of melanoma and potentially other cancers.

[0527] Based upon the observed homology, the polypeptide of the presentinvention is expected to share at least some biological activity withother transient receptor potential channel family members, morespecifically with the melastatin1 protein, in addition to, othertransient receptor potential channel family members referenced elsewhereherein or otherwise known in the art, particularly LTRPC3f, and LTRPC3.

[0528] Most of the known transient receptor potential channel familymembers, possess one or more transmembrane domains. Likewise, theLTRPC3l polypeptide has been determined to comprise six transmembranedomains (TM1-TM6) as shown in FIGS. 6A-F. The transmembrane domains arelocated from about amino acid 800 to about amino acid 817 (TM1), fromabout amino acid 897 to about amino acid 914 (TM2), from about aminoacid 970 to about amino acid 983 (TM3), from about amino acid 997 toabout amino acid 1014 (TM4), from about amino acid 1031 to about aminoacid 1048 (TM5), and/or from about amino acid 1118 to about amino acid1137 (TM6) of SEQ ID NO:12. In this context, the term “about” may beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0529] In preferred embodiments, the following transmembrane domainpolypeptides are encompassed by the present invention:LKVILGILLPPSILSLEF (SEQ ID NO:92), IVKFWFYTLAYIGYLMLF (SEQ ID NO:93),VTDLIAILLFSVGM (SEQ ID NO:94), RVIYCVNIIYWYIRLLDI (SEQ ID NO:95),MMIDMMYFVIIMLVVLMS (SEQ ID NO:96), and/or AIMACYLLVANILLVNLLIAV (SEQ IDNO:97). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of the LTRPC3ltransmembrane polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

[0530] The present invention also encompasses polypeptides correspondingto the regions between each transmembrane domain of LTRPC3l, referred toherein as inter-transmembrane domain polypeptides (inter TM1-2, interTM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions aretypically solvent accessible (either extracellularly orintracellularly). Therefore, these regions are amenable to thedevelopment of agonists and/or antagonists to the LTRPC3l polypeptide,particularly for the development of antibodies specific to theseregions.

[0531] In preferred embodiments, the following inter-transmembranedomain polypeptides are encompassed by the present invention:KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESSRKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:98),NYIVLVKMERWPSTQEWIVISYFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:99),ILRLQDQPFRSDG (SEQ ID NO:100), FGVNKYLGPYVMMIGK (SEQ ID NO:101), and/orFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDRKQVYDSHTPKSAPCGQNETREDGKIIQLPPCKTGAWIVP (SEQ ID NO:102). Polynucleotides encodingthese polypeptides are also provided. The present invention alsoencompasses the use of the LTRPC3l inter-transmembrane polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

[0532] The LTRPC3l polypeptide was determined to comprise severalconserved cysteines, at amino acid 249, 389, 599, 697, 762, 779, 1001,1092, 1109, 1122, 1225, 1316, and 1582 of SEQ ID No: 12 (FIGS. 6A-F).Conservation of cysteines at key amino acid residues is indicative ofconserved structural features, which may correlate with conservation ofprotein function and/or activity.

[0533] In confirmation of the LTRPC3l representing a member of thetransient receptor channel family, the LTRPC3l polypeptide wasdetermined to comprise a predicted TRP domain located from about aminoacid 1153 to about amino acid 1158 of SEQ ID NO:12. In this context, theterm “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids beyond the N-Terminus and/or C-terminus of the abovereferenced polypeptide.

[0534] In preferred embodiments, the following TRP domain polypeptide isencompassed by the present invention: VWKFQR (SEQ ID NO:103).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3l TRP domainpolypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

[0535] In further confirmation of the LTRPC3l representing a member ofthe transient receptor channel family, the LTRPC3l polypeptide wasdetermined to comprise a predicted ion transport signature domainlocated at about amino acid 928 to about amino acid 1136 of SEQ IDNO:12. In this context, the term “about” may be construed to mean 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/orC-terminus of the above referenced polypeptide.

[0536] In preferred embodiments, the following ion transport signaturedomain polypeptide is encompassed by the present invention:TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:104).Polynucleotides encoding this polypeptide are also provided. The presentinvention also encompasses the use of this LTRPC3l ion transportsignature domain polypeptide as an immunogenic and/or antigenic epitopeas described elsewhere herein.

[0537] The LTRPC3l polypeptide was determined to comprise a predictedpredicted coiled-coil domain located at about amino acid 1243 to aboutamino acid 1298 of SEQ ID NO:12. In this context, the term “about” maybe construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyondthe N-Terminus and/or C-terminus of the above referenced polypeptide.

[0538] In preferred embodiments, the following coiled-coil domainpolypeptide is encompassed by the present invention:ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ IDNO:105). Polynucleotides encoding this polypeptide are also provided.The present invention also encompasses the use of this LTRPC3lcoiled-coil domain polypeptide as an immunogenic and/or antigenicepitope as described elsewhere herein.

[0539] LTRPC3l polypeptides and polynucleotides are useful fordiagnosing diseases related to the over and/or under expression ofLTRPC3l by identifying mutations in the LTRPC3l gene using LTRPC3lsequences as probes or by determining LTRPC3l protein or mRNA expressionlevels. LTRPC3l polypeptides will be useful in screens for compoundsthat affect the activity of the protein. LTRPC3l peptides can also beused for the generation of specific antibodies and as bait in yeast twohybrid screens to find proteins the specifically interact with LTRPC3l.

[0540] Since the LTRPC3l polypeptide represents an N-terminally extendedvariant of the LTRPC3f polypeptide (see polypeptide alignment providedin FIG. 11A-I), and since the LTRPC3f (SEQ ID NO:21) polypeptiderepesents a splice variant of the LTRPC3 polypeptide (SEQ ID NO:15), theLTRPC3l (SEQ ID NO:12) polypeptide of the present invention is expectedto have the same or similar function as the LTRPC3 polypeptide, and inparticular, the LTRPC3f polypeptide. Specifically, the LTRPC3lpolypeptide is expected to localize to the cell membrane.

[0541] Moreover, the LTRPC3l polypeptide is expected to also localize inor near the plasmalemmal compartment.

[0542] Moreover, the LTRPC3l polypeptide is also expected to be aconstitutively active channel capable of mediating Ca²⁺ influx.

[0543] Moreover, the LTRPC3l polypeptide is also expected to mediateCa²⁺ entry and that LTRPC3l-mediated Ca²⁺ entry is expected to bepotentiated by store-depletion.

[0544] Moreover, the LTRPC3l polypeptide is also expected to mediate aCa²⁺ entry pathway that is distinct from the endogenous Ca²⁺ entrypathways present in HEK 293 cells.

[0545] Moreover, the LTRPC3l polypeptide of the present invention isexpected to share the same or similar expression profile as that of theLTRPC3 polypeptide (SEQ ID NO:15).

[0546] Moreover, the LTRPC3l polypeptide is expected to share the sameor similar differential expression patterns observed for the LTRPC3variant.

[0547] Moreover, the LTRCP3g polypeptide is expected to be able tomodulate the FEN1 DNA base-excision repair/proliferation modulatingprotein to the same or similar extent as the LTRPC3 polypeptide.

[0548] Moreover, the LTRPC3l polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in kidney cells and tissues, particularly thosespecifically referenced herein, such as LTRPC3 (SEQ ID NO:15), andparticularly as LTRPC3b (SEQ ID NO:17).

[0549] Moreover, the LTRPC3l polynucleotides and polypeptides areexpected to share at least some biological activity with TRP familymembers expressed in testis cells and tissues, particularly thosespecifically referenced herein, such as the LTRPC3 variant (SEQ IDNO:15), and particularly as LTRPC3f (SEQ ID NO:21).

[0550] In preferred embodiments, LTRPC3l polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating Ca²⁺ reabsorption disorders,disorders associated with aberrant luminal Ca2+ entry via the epithelialCa2+ channel (ECaC), disorders associated with aberrant cytosolicdiffusion of Ca2+ bound to calbindin-D28K, disorders associated withaberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger(NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase(PMCA) activity and/or activation, disorders associated with thehypocalciuric effect of diuretics, disorders associated with thehypocalciuric effect of thiazide diuretics, disorders associated withhormone insufficiencies that affect the function of the kidney,disorders associated with hormone insufficiencies that affect renal Ca2+homeostasis, disorders associated with aberrant renal Ca2+ homeostasisas a result of vitamin D deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of PTH deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofvasopressin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of prostaglandines deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of estrogendeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of calcitonin deficiency, disorders associated with aberrantrenal Ca2+ homeostasis as a result of parathyroid hormone deficiency,disorders associated with aberrant renal Ca2+ homeostasis as a result ofatrial natriuretic peptide deficiency, calcium homeostasis-relateddisorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis,Gitelman's disease, Bartter's syndrom, disorders associated withaberrant function of kidney tubular epithelium, disorders associatedwith aberrant function of kidney tubular epithelium in the medulla,disorders associated with aberrant function of kidney tubular epitheliumin the medullary rays, disorders associated with aberrant function ofkidney tubular epithelium in the periglomerular.

[0551] In preferred embodiments, LTRPC3l polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.

[0552] Characterization of the LTRPC3 variant polypeptide usingantisense oligonucleotides led to the determination that LTRPC3 isinvolved in the negative modulation of the FEN1 DNA base-excisionrepair/proliferation modulating protein as described in Example 5herein.

[0553] The LTRCP3g polypeptide is expected to be able to modulate theFEN1 DNA base-excision repair/proliferation modulating protein to thesame or similar extent as the LTRPC3 polypeptide.

[0554] Specifically, antisense inhibition of LTRPC3 resulted inupregulation of Fen1, a marker of proliferation. As such, LTRPC3 behavesin a manner similar to a tumor suppressor, in that loss results inincreases in proliferation. While some increases in the proliferationmarkers p21 and IkB were also observed, they were less significant, andcould be a response to the pro-proliferative effect of LTRPC3 antisenseinhibition. Interestingly, expression of LTRPC3 was also observed to besignificantly reduced in tumor tissues, relative to normal tissues (seeFIG. 13). Specifically, renal, testicular, and ovarian tumor samplesshowed as much as 12 to 30 fold down regulation of LTRPC3 message levelscompared to normal tissues. This reduction of LTRPC3 correlating withthe tumor state is consistent with the negative FEN1modulatory activityand suggests this gene may have antiproliferative activities. Therefore,for modulators of LTRPC3l to be useful for treating cancer, it would benecessary to increase the activity of the gene or gene product in orderto have the reverse effect on cancerous cells. Thus, an agonist ofLTRPC3l activity or a compound that increased LTRPC3l message levelswould be a desired invention for cancer therapy.

[0555] In preferred embodiments, LTRPC3l polynucleotides andpolypeptides, including fragments and modulators thereof, are useful fortreating, diagnosing, and/or ameliorating DNA-repair deficiencies,particularly base-excision repair deficiencies, Xeroderma pigmentosum,skin cancer, melanoma, UV senstivity, alkylation sensivity, gammairradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0556] Moreover, LTRPC3l polynucleotides and polypeptides, includingfragments and modulators thereof, are useful for increasing mammalianbase excision repair activity, increasing mammalian single-nucleotidebase excision repair activity, and/or increasing mammalian long patchbase excision repair activity.

[0557] In preferred embodiments, antagonists directed against LTRPC3lare useful for treating, diagnosing, and/or ameliorating DNA-repairdeficiencies, particularly base-excision repair deficiencies, Xerodermapigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity,gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemicalmutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone,Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, bloodvessel dilation aberrations in the skin, blood vessel dilationaberrations in the eye, conditions involving increased levels ofapurinic sites, conditions involving increased levels of apyrimidinicsites, conditions involving increased levels of abasic sites, disordersrelated to aberrant signal transduction, proliferating disorders, and/orcancers.

[0558] Moreover, antagonists directed against LTRPC3l are useful forincreasing mammalian base excision repair activity, increasing mammaliansingle-nucleotide base excision repair activity, and/or increasingmammalian long patch base excision repair activity.

[0559] In preferred embodiments, agonists directed against LTRPC3l areuseful for treating, diagnosing, and/or ameliorating, disorders relatedto aberrant signal transduction, proliferating disorders, and/orcancers, particularly renal cell carcinomas, testicular cancers, and/orovarian cancers.

[0560] Moreover, agonists directed against LTRPC3l are useful fordecreasing mammalian base excision repair activity, decreasing mammaliansingle-nucleotide base excision repair activity, and/or decreasingmammalian long patch base excision repair activity.

[0561] The strong homology to human transient receptor potentialchannels (TRP), combined with the predominate localized expression inkidney tissue of the LTRPC3 variant, suggests the LTRPC3lpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing renal diseases and/or disorders, which include, but are notlimited to: nephritis, renal failure, nephrotic syndrome, urinary tractinfection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema,hypertension, electrolyte disorders, sterile pyuria, renalosteodystrophy, large kidneys, renal transport defects, nephrolithiasis,azotemia, anuria, urinary retention, slowing of urinary stream, largeprostate, flank tenderness, full bladder sensation after voiding,enuresis, dysuria,bacteriuria, kideny stones, glomerulonephritis,vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenicpurpura, malignant hypertension, casts, tubulointerstitial kidneydiseases, renal tubular acidosis, pyelonephritis, hydronephritis,nephrotic syndrome, crush syndrome, and/or renal colic, in addition toWilm's Tumor Disease, and congenital kidney abnormalities such ashorseshoe kidney, polycystic kidney, and Falconi's syndrome for example.

[0562] Several known TRP family members have been identified that areexpressed significantly in kidney tissue. These TRP family membersinclude, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M.,Flockerzi, V, Lett., 485(2-3): 127-34, (2000)); OTRPC4 (Strotmann, R.,Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell,Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A,W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H.,Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).

[0563] Thus, the LTRPC3l polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in kidney cells and tissues, particularly those specificallyreferenced herein, such as LTRPC3 (SEQ ID NO:15).

[0564] The strong homology to human human transient receptor potentialchannels (TRP) proteins, combined with the localized expression inspinal cord and brain of the LTRPC3 variant, suggests the LTRPC3lpolynucleotides and polypeptides, including modulators or fragmentsthereof, may be useful in treating, diagnosing, prognosing, and/orpreventing neurodegenerative disease states, behavioral disorders, orinflammatory conditions. Representative uses are described in the“Regeneration” and “Hyperproliferative Disorders” sections below, in theExamples, and elsewhere herein. Briefly, the uses include, but are notlimited to the detection, treatment, and/or prevention of Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,meningitis, encephalitis, demyelinating diseases, peripheralneuropathies, neoplasia, trauma, congenital malformations, spinal cordinjuries, ischemia and infarction, aneurysms, hemorrhages,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,depression, panic disorder, learning disabilities, ALS, psychoses,autism, and altered behaviors, including disorders in feeding, sleeppatterns, balance, and perception. In addition, elevated expression ofthis gene product in regions of the brain indicates it plays a role innormal neural function. Potentially, this gene product is involved insynapse formation, neurotransmission, learning, cognition, homeostasis,or neuronal differentiation or survival. Furthermore, the protein mayalso be used to determine biological activity, to raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

[0565] The strong homology to human transient receptor potentialchannels (TRP), combined with the localized expression in testis tissueof the LTRPC3 variant emphasizes the potential utility for LTRPC3lpolynucleotides and polypeptides in treating, diagnosing, prognosing,and/or preventing testicular, in addition to reproductive disorders.

[0566] In preferred embodiments, LTRPC3l polynucleotides andpolypeptides including agonists and fragments thereof, have uses whichinclude treating, diagnosing, prognosing, and/or preventing thefollowing, non-limiting, diseases or disorders of the testis:spermatogenesis, infertility, Klinefelter's syndrome, XX male,epididymitis, genital warts, germinal cell aplasia, cryptorchidism,varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3lpolynucleotides and polypeptides including agonists and fragmentsthereof, may also have uses related to modulating testiculardevelopment, embryogenesis, reproduction, and in ameliorating, treating,and/or preventing testicular proliferative disorders (e.g., cancers,which include, for example, choriocarcinoma, Nonseminoma, seminona, andtesticular germ cell tumors).

[0567] Likewise, the localized expression in testis tissue alsoemphasizes the potential utility for LTRPC3l polynucleotides andpolypeptides in treating, diagnosing, prognosing, and/or preventingmetabolic diseases and disorders which include the following, notlimiting examples: premature puberty, incomplete puberty, Kallmansyndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis,congenital adrenal hyperplasia, FSH deficiency, and granulomatousdisease, for example.

[0568] This gene product may also be useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

[0569] Several known TRP family members have been identified that areexpressed significantly in testis tissue. These TRP family membersinclude, for example, polycystin-L2 (Guo, L., Schreiber, T, H.,Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics.,64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda,A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K.,Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2(Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett.,429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M.,Birnbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D.,Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C,Proc, Natl, Acad, Sci, U, S, A., 92(21):9652-6, (1995)).

[0570] Thus, the LTRPC3l polynucleotides and polypeptides are expectedto share at least some biological activity with TRP family membersexpressed in testis cells and tissues, particularly those specificallyreferenced herein, such as the LTRPC3 variant (SEQ ID NO:15).

[0571] As described elsewhere herein, transient receptor potentialchannel family members have been implicated in modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity. Moreover, transient receptorpotential channel family members have been implicated in disorders ofthe skin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0572] In preferred embodiments, LTRPC3l polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, modulating cellproliferation, differentiation, migration, activation, exocytosis,muscle contraction, gene expression, apoptosis, signalling, pheromonesensory signaling, smooth muscle tone, pain perception, heat perception,osmosenstivity, and mechanosensitivity.

[0573] In more preferred embodiments, LTRPC3l polynucleotides andpolypeptides of the present invention, including agonists and/orfragments thereof, have uses that include, treating, ameliorating,preventing, detecting, and/or prognosing various diseases and disorders,particularly the following, non-limiting examples, disorders of theskin, skeletal-muscle, nervous, cardiac, and vascular systems, inaddition to the following, non-limiting diseases and disorders, whichinclude, for example, arteriosclerosis, neointimal hypoerplasia,metastatic melanomas, bipolar disorder, nonsyndromic hereditarydeafness, Knobloch syndrome, holosencephaly, and various maliganciesincluding prostate cancer.

[0574] LTRPC3l polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments may be involved inintracellular Ca²⁺ homeostasis which affects various aspects ofbiological functions including mechano-regulation, pain transduction,vasorelaxation, gene expression, cell cycle and proliferation/apoptosis.Since the LTRPC3 variant is dominantly expressed in kidney, the LTRPC3lpolypeptide may particularly play an important role in regulatingcytosolic Ca2+ in the renal system, potentially Ca2+ absorption.

[0575] The LTRPC3l gene maps to chromosome 9q21.11-21.31 between the twomarkers D9S1874 and D9S1807. This region is linked to amyotrophiclateral sclerosis with frontotemporal dementia, early-onset pulverulentcataract, infantile nephronophthisis, hypomagnesemia with secondaryhypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis.Therefore, agonists and/or antagonists of the novel LTRPC3l can be usedto treat diseases including various forms of neuronal degeneration,neurogenic inflammation, allergy, immunodeficiency/excessive immuneactivation, visual defects, hearing disorder, pain, cancer, hypertensionand other cardiovascular diseases. In addition, the therapeutics may beuseful in the treatment of diseases associated with disturbances in Ca²⁺homeostasis including osteoporosis, hypercalciuric stone disease, andchronic renal failure.

[0576] Moreover, given the selective expression in kidney, LTRPC3l couldbe considered a candidate gene for HSH, since the phenotype is a renalinsufficiency. LTRPC3l chromosomal location is ˜600 kb downstream of aX;9 translocation breakpoint interval described for one patient with HSH(Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reportedthat a new member of the TRPM subfamily, TRPM6, expressed in bothintestinal tissues and kidney, is associated with HSH (Nat. Genet. 31,166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3l is˜4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first componentidentified for HSH, this should not preclude involvement of LTRPC3l inthe disease. Therefore, it is possible that LTRPC3l may co-localize withTRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.

[0577] The kidney plays a major role in Ca²⁺ homeostasis (reviewed inPhysiol. Rev. 75, 429-471, (1995)). LTRPC3l may play a direct role inCa²⁺ absorption due to its constitutive Ca²⁺ permeability.Alternatively, LTRPC3l may be the SOC that regulates Ca²⁺ absorption. Inkidney, Ca²⁺ absorption is regulated by agonists such as calcitonin,parathyroid hormone and parathyroid hormone-related peptide throughtheir respective G protein-coupled receptors, and downstream SOCs.Additionally, in kidney a SOC has been described (J. Biol. Chem. 276,25759-25765, (2001)) for the action of angiotensin II, a vasoactivepeptide that plays a major role in regulating blood pressure.

[0578] In addition, LTRPC3l polynucleotides and polypeptides of thepresent invention, including agonists and/or fragments thereof, haveuses that include modulating intracellular Ca++ ion concentrations, Ca++ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pumpactivity, Ca++ ion flow into cell, Ca++ ion flow out of cells, theactivation of Ca++ sensitive proteins, the activation of Ca++ sensitivesignaling pathways, the activation of kinase-activatible proteins, andthe activation of kinase-dependent signaling pathways.

[0579] The LTRPC3l polynucleotides and polypeptides of the presentinvention, including agonists and/or fragments thereof, have uses thatinclude modulating proliferation, differentiation, migration, andactivation in various cells, tissues, and organisms, and particularly inmammalian kidney, spinal cord, testis, and brain, preferably human.LTRPC3l polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, may be useful indiagnosing, treating, prognosing, and/or preventing renal, neural,and/or proliferative diseases or disorders, particularly of the renalsystem.

[0580] In addition, antagonists of the LTRPC3l polynucleotides andpolypeptides, including modulators or fragments thereof, may have usesthat include diagnosing, treating, prognosing, and/or preventingdiseases or disorders related to transient receptor potential channelactivity, which may include renal, neural, reproductive, and/orproliferative diseases or disorders.

[0581] Although it is believed the encoded polypeptide may share atleast some biological activities with transient receptor potentialchannel family members, particularly those melastatin1, a number ofmethods of determining the exact biological function of this clone areeither known in the art or are described elsewhere herein. Briefly, thefunction of this clone may be determined by applying microarraymethodology. Nucleic acids corresponding to the LTRPC3l polynucleotides,in addition to, other clones of the present invention, may be arrayed onmicrochips for expression profiling. Depending on which polynucleotideprobe is used to hybridize to the slides, a change in expression of aspecific gene may provide additional insight into the function of thisgene based upon the conditions being studied. For example, an observedincrease or decrease in expression levels when the polynucleotide probeused comes from tissue that has been treated with known transientpotential receptor inhibitors, which include, but are not limited to thedrugs listed herein or otherwise known in the art, might indicate afunction in modulating transient potential receptor function, forexample. In the case of LTRPC3l, kidney, spinal cord, testis, and brain,should be used to extract RNA to prepare the probe.

[0582] In addition, the function of the protein may be assessed byapplying quantitative PCR methodology, for example. Real timequantitative PCR would provide the capability of following theexpression of the LTRPC3l gene throughout development, for example.Quantitative PCR methodology requires only a nominal amount of tissuefrom each developmentally important step is needed to perform suchexperiements. Therefore, the application of quantitative PCR methodologyto refining the biological function of this polypeptide is encompassedby the present invention. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-E).

[0583] The function of the protein may also be assessed throughcomplementation assays in yeast. For example, in the case of theLTRPC3l, transforming yeast deficient in transient receptor potentialchannel activity with LTRPC3l and assessing their ability to grow wouldprovide convincing evidence the LTRPC3l polypeptide has transientreceptor potential channel activity. Additional assay conditions andmethods that may be used in assessing the function of the polynucletidesand polypeptides of the present invention are known in the art, some ofwhich are disclosed elsewhere herein.

[0584] Alternatively, the biological function of the encoded polypeptidemay be determined by disrupting a homologue of this polypeptide in Miceand/or rats and observing the resulting phenotype.

[0585] Moreover, the biological function of this polypeptide may bedetermined by the application of antisense and/or sense methodology andthe resulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the obervation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a kidney, spinalcord, testis, and/or brain-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

[0586] In the case of LTRPC3l transgenic mice or rats, if no phenotypeis apparent in normal growth conditions, observing the organism underdiseased conditions (renal, reproductive, neural, or proliferativedisorders, etc.) may lead to understanding the function of the gene.Therefore, the application of antisense and/or sense methodology to thecreation of transgenic mice or rats to refine the biological function ofthe polypeptide is encompassed by the present invention.

[0587] In preferred embodiments, the following N-terminal LTRPC3ldeletion polypeptides are encompassed by the present invention:M1-T1734, G2-T1734, K3-T1734, K4-T1734, W5-T1734, R6-T1734, D7-T1734,A8-T1734, A9-T1734, E10-T1734, M11-T1734, E12-T1734, R13-T1734,G14-T1734, C15-T1734, S16-T1734, D17-T1734, R18-T1734, E19-T1734,D20-T1734, N21-T1734, A22-T1734, E23-T1734, S24-T1734, R25-T1734,R26-T1734, R27-T1734, S28-T1734, R29-T1734, S30-T1734, A31-T1734,S32-T1734, R33-T1734, G34-T1734, R35-T1734, F36-T1734, A37-T1734,E38-T1734, S39-T1734, W40-T1734, K41-T1734, R42-T1734, L43-T1734,S44-T1734, S45-T1734, K46-T1734, Q47-T1734, G48-T1734, S49-T1734,T50-T1734, K51-T1734, R52-T1734, S53-T1734, G54-T1734, L55-T1734,P56-T1734, S57-T1734, Q58-T1734, Q59-T1734, T60-T1734, P61-T1734,A62-T1734, Q63-T1734, K64-T1734, S65-T1734, W66-T1734, I67-T1734,E68-T1734, R69-T1734, A70-T1734, F71-T1734, Y72-T1734, K73-T1734,R74-T1734, E75-T1734, C76-T1734, V77-T1734, H78-T1734, I79-T1734,I80-T1734, P81-T1734, S82-T1734, T83-T1734, K84-T1734, D85-T1734,P86-T1734, H87-T1734, R88-T1734, C89-T1734, C90-T1734, C91-T1734,G92-T1734, R93-T1734, L94-T1734, I95-T1734, G96-T1734, Q97-T1734,H98-T1734, V99-T1734, G100-T1734, L101-T1734, T102-T1734, P103-T1734,S104-T1734, I105-T1734, S106-T1734, V107-T1734, L108-T1734, Q109-T1734,N110-T1734, E111-T1734, K112-T1734, N113-T1734, E114-T1734, S115-T1734,R116-T1734, L117-T1734, S118-T1734, R119-T1734, N120-T1734, D121-T1734,I122-T1734, Q123-T1734, S124-T1734, E125-T1734, K126-T1734, W127-T1734,S128-T1734, I129-T1734, S130-T1734, K131-T1734, H132-T1734, T133-T1734,Q134-T1734, L135-T1734, S136-T1734, P137-T1734, T138-T1734, D139-T1734,A140-T1734, F141-T1734, G142-T1734, T143-T1734, I144-T1734, E145-T1734,F146-T1734, Q147-T1734, G148-T1734, G149-T1734, G150-T1734, H151-T1734,S152-T1734, N153-T1734, K154-T1734, and/or A155-T1734 of SEQ ID NO:12.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalLTRPC3l deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

[0588] In preferred embodiments, the following C-terminal LTRPC3ldeletion polypeptides are encompassed by the present invention:M1-T1734, M1-H1733, M1-K1732, M1-S1731, M1-E1730, M1-F1729, M1-S1728,M1-Q1727, M1-F1726, M1-A1725, M1-S1724, M1-T1723, M1-R1722, M1-S1721,M1-L1720, M1-R1719, M1-R1718, M1-M1717, M1-S1716, M1-L1715, M1-S1714,M1-D1713, M1-G1712, M1-R1711, M1-G1710, M1-E1709, M1-P1708, M1-K1707,M1-S1706, M1-S1705, M1-K1704, M1-S1703, M1-R1702, M1-Q1701, M1-F1700,M1-P1699, M1-N1698, M1-Q1697, M1-L1696, M1-S1695, M1-A1694, M1-T1693,M1-N1692, M1-R1691, M1-Q1690, M1-R1689, M1-D1688, M1-L1687, M1-K1686,M1-D1685, M1-S1684, M1-I1683, M1-S1682, M1-F1681, M1-S1680, M1-K1679,M1-R1678, M1-T1677, M1-H1676, M1-A1675, M1-Y1674, M1-P1673, M1-A1672,M1-S1671, M1-P1670, M1-E1669, M1-E1668, M1-A1667, M1-S1666, M1-Y1665,M1-S1664, M1-N1663, M1-A1662, M1-R1661, M1-E1660, M1-I1659, M1-K1658,M1-P1657, M1-V1656, M1-T1655, M1-I1654, M1-N1653, M1-N1652, M1-S1651,M1-L1650, M1-T1649, M1-R1648, M1-E1647, M1-S1646, M1-N1645, M1-D1644,M1-G1643, M1-E1642, M1-Q1641, M1-S1640, M1-S1639, M1-I1638, M1-A1637,M1-I1636, M1-T1635, M1-A1634, M1-R1633, M1-R1632, M1-G1631, M1-K1630,M1-A1629, M1-E1628, M1-N1627, M1-E1626, M1-E1625, M1-S1624, M1-D1623,M1-S1622, M1-S1621, M1-P1620, M1-H1619, M1-S1618, M1-L1617, M1-E1616,M1-A1615, M1-E1614, M1-R1613, M1-E1612, M1-P1611, M1-H1610, M1-C1609,M1-C1608, M1-T1607, M1-L1606, M1-D1605, M1-E1604, M1-V1603, M1-K1602,M1-D1601, M1-G1600, M1-L1599, M1-G1598, M1-G1597, M1-P1596, M1-F1595,M1-A1594, M1-A1593, M1-R1592, M1-D1591, M1-A1590, M1-I1589, M1-A1588,M1-Q1587, M1-P1586, M1-A1585, M1-N1584, M1-V1583, M1-C1582, M1-R1581,M1-T1580, M1-D1579, M1-I1578, M1-C1577, M1-D1576, M1-T1575, M1-I1574,M1-S1573, M1-T1572, M1-Y1571, M1-E1570, M1-A1569, M1-T1568, M1-K1567,M1-V1566, M1-P1565, M1-V1564, M1-G1563, M1-F1562, M1-N1561, M1-A1560,M1-Y1559, M1-Y1558, M1-S1557, M1-R1556, M1-S1555, M1-P1554, M1-S1553,M1-F1552, M1-M1551, M1-F1550, M1-S1549, M1-H1548, M1-S1547, M1-K1546,M1-V1545, M1-I1544, M1-P1543, M1-A1542, M1-E1541, M1-E1540, M1-L1539,M1-L1538, M1-F1537, M1-P1536, M1-T1535, M1-T1534, M1-A1533, M1-L1532,M1-Y1531, M1-R1530, M1-S1529, M1-S1528, M1-K1527, M1-S1526, M1-R1525,M1-E1524, M1-I1523, M1-T1522, M1-H1521, M1-Y1520, M1-M1519, M1-P1518,M1-P1517, M1-E1516, M1-S1515, M1-D1514, M1-W1513, M1-P1512, M1-N1511,M1-Q1510, M1-C1509, M1-E1508, M1-P1507, M1-L1506, M1-H1505, M1-T1504,M1-Y1503, M1-D1502, M1-S1501, M1-S1500, M1-F1499, M1-S1498, M1-R1497,M1-T1496, M1-D1495, M1-M1494, M1-S1493, M1-T1492, M1-I1491, M1-D1490,M1-E1489, M1-F1488, M1-D1487, M1-I1486, M1-S1485, M1-R1484, M1-S1483,M1-P1482, M1-P1481, M1-R1480, M1-D1479, M1-T1478, M1-P1477, M1-A1476,M1-L1475, M1-T1474, M1-A1473, M1-Y1472, M1-A1471, M1-S1470, M1-S1469,M1-S1468, M1-P1467, M1-A1466, M1-T1465, M1-S1464, M1-P1463, M1-V1462,M1-P1461, M1-T1460, M1-S1459, M1-F1458, M1-S1457, M1-P1456, M1-E1455,M1-G1454, M1-L1453, M1-G1452, M1-L1451, M1-I1450, M1-N1449, M1-V1448,M1-S1447, M1-N1446, M1-D1445, M1-L1444, M1-P1443, M1-D1442, M1-I1441,M1-D1440, M1-C1439, M1-H1438, M1-L1437, M1-E1436, M1-D1435, M1-M1434,M1-A1433, M1-S1432, M1-V1431, M1-Y1430, M1-I1429, M1-D1428, M1-I1427,M1-C1426, M1-S1425, M1-S1424, M1-P1423, M1-R1422, M1-R1421, M1-S1420,M1-D1419, M1-P1418, M1-V1417, M1-I1416, M1-A1415, M1-L1414, M1-T1413,M1-N1412, M1-A1411, M1-P1410, M1-A1409, M1-A1408, M1-P1407, M1-A1406,M1-K1405, M1-P1404, M1-E1403, M1-K1402, M1-A1401, M1-V1400, M1-S1399,M1-H1398, M1-S1397, M1-S1396, M1-T1395, M1-A1394, M1-R1393, M1-H1392,M1-L1391, M1-S1390, M1-L1389, M1-S1388, M1-R1387, M1-E1386, M1-K1385,M1-F1384, M1-I1383, M1-S1382, M1-E1381, M1-L1380, M1-K1379, M1-E1378,M1-I1377, M1-G1376, M1-G1375, M1-K1374, M1-D1373, M1-K1372, M1-M1371,M1-N1370, M1-V1369, M1-S1368, M1-Y1367, M1-F1366, M1-S1365, M1-H1364,M1-S1363, M1-R1362, M1-M1361, M1-R1360, M1-P1359, M1-M1358, M1-L1357,M1-T1356, M1-P1355, M1-S1354, M1-T1353, M1-P1352, M1-S1351, M1-M1350,M1-T1349, M1-E1348, M1-E1347, M1-G1346, M1-A1345, M1-P1344, M1-D1343,M1-I1342, M1-S1341, M1-E1340, M1-Q1339, M1-L1338, M1-K1337, M1-F1336,M1-T1335, M1-N1334, M1-G1333, M1-E1332, M1-Q1331, M1-S1330, M1-N1329,M1-F1328, M1-S1327, M1-S1326, M1-Q1325, M1-R1324, M1-V1323, M1-I1322,M1-Y1321, M1-A1320, M1-A1319, M1-D1318, M1-T1317, M1-C1316, M1-D1315,M1-S1314, M1-S1313, M1-T1312, M1-R1311, M1-S1310, M1-R1309, M1-I1308,M1-K1307, M1-N1306, M1-S1305, M1-E1304, M1-A1303, M1-R1302, M1-E1301,M1-L1300, M1-G1299, M1-T1298, M1-L1297, M1-R1296, M1-E1295, M1-L1294,M1-A1293, M1-T1292, M1-A1291, M1-M1290, M1-R1289, M1-G1288, M1-I1287,M1-L1286, M1-D1285, M1-E1284, M1-L1283, M1-Q1282, M1-A1281, M1-L1280,M1-R1279, M1-I1278, M1-D1277, M1-V1276, M1-T1275, M1-Q1274, M1-L1273,M1-S1272, M1-A1271, M1-K1270, M1-M1269, M1-S1268, M1-H1267, M1-E1266,M1-R1265, M1-E1264, M1-N1263, M1-V1262, M1-E1261, M1-E1260, M1-L1259,M1-R1258, M1-M1257, M1-S1256, M1-M1255, M1-N1254, M1-E1253, M1-V1252,M1-R1251, M1-E1250, M1-S1249, M1-T1248, M1-V1247, M1-R1246, M1-I1245,M1-R1244, M1-E1243, M1-D1242, M1-N1241, M1-S1240, M1-S1239, M1-N1238,M1-F1237, M1-R1236, M1-D1235, M1-D1234, M1-K1233, M1-E1232, M1-R1231,M1-F1230, M1-Y1229, M1-E1228, M1-E1227, M1-I1226, M1-C1225, M1-Q1224,M1-E1223, M1-E1222, M1-F1221, M1-D1220, M1-H1219, M1-V1218, M1-K1217,M1-K1216, M1-L1215, M1-E1214, M1-D1213, M1-D1212, M1-T1211, M1-I1210,M1-F1209, M1-L1208, M1-K1207, M1-L1206, M1-G1205, M1-Y1204, M1-D1203,M1-R1202, M1-E1201, M1-D1200, M1-P1199, M1-D1198, M1-S1197, M1-E1196,M1-H1195, M1-K1194, M1-R1193, M1-W1192, M1-R1191, M1-C1190, M1-C1189,M1-L1188, M1-H1187, M1-Q1186, M1-F1185, M1-I1184, M1-M1183, M1-T1182,M1-M1181, M1-H1180, M1-S1179, M1-F1178, M1-I1177, M1-I1176, M1-L1175,M1-P1174, M1-P1173, M1-P1172, M1-L1171, M1-V1170, M1-P1169, M1-R1168,M1-E1167, M1-H1166, M1-F1165, M1-T1164, M1-M1163, M1-I1162, M1-L1161,M1-Q1160, M1-Y1159, M1-R1158, M1-Q1157, M1-F1156, M1-K1155, M1-W1154,M1-V1153, M1-Q1152, M1-N1151, M1-S1150, M1-I1149, M1-S1148, M1-K1147,M1-V1146, M1-E1145, M1-F1144, M1-F1143, M1-T1142, M1-N1141, M1-N1140,M1-F1139, M1-V1138, M1-A1137, M1-I1136, M1-L1135, M1-L1134, M1-N1133,M1-V1132, M1-L1131, M1-L1130, M1-I1129, M1-N1128, M1-A1127, M1-V1126,M1-L1125, M1-L1124, M1-Y1123, M1-C1122, M1-A1121, M1-M1120, M1-I1119,M1-A 1118, M1-P1117, M1-V1116, M1-I1115, M1-W1114, M1-A1113, M1-G1112,M1-T1111, M1-K1110, M1-C1109, M1-P1108, M1-P1107, M1-L1106, M1-Q 105,M1-I1104, M1-I1103, M1-K1102, M1-G1101, M1-D1100, M1-E1099, M1-R1098,M1-T1097, M1-E1096, M1-N1095, M1-Q1094, M1-G1093, M1-C1092, M1-P1091,M1-P1090, M1-D1089, M1-I1088, M1-Q1087, M1-D1086, M1-A1085, M1-F1084,M1-V1083, M1-E1082, M1-G1081, M1-Y1080, M1-I1079, M1-M1078, M1-W1077,M1-Y1076, M1-P1075, M1-M1074, M1-Y1073, M1-F1072, M1-I1071, M1-N1070,M1-K1069, M1-A1068, M1-L1067, M1-K1066, M1-W1065, M1-S1064, M1-P1063,M1-E1062, M1-E1061, M1-N1060, M1-P1059, M1-F1058, M1-L1057, M1-I1056,M1-A1055, M1-Q1054, M1-R1053, M1-A1052, M1-V1051, M1-G1050, M1-F1049,M1-S1048, M1-M1047, M1-L1046, M1-V1045, M1-V1044, M1-L1043, M1-M1042,M1-I1041, M1-I1040, M1-V1039, M1-F1038, M1-Y1037, M1-M1036, M1-M1035,M1-D1034, M1-I1033, M1-M1032, M1-M1031, M1-K1030, M1-G1029, M1-I1028,M1-M1027, M1-M1026, M1-V1025, M1-Y1024, M1-P1023, M1-G1022, M1-L1021,M1-Y1020, M1-K1019, M1-N1018, M1-V1017, M1-G1016, M1-F1015, M1-I1014,M1-D1013, M1-L1012, M1-L1011, M1-R1010, M1-I1009, M1-Y1008, M1-W1007,M1-Y1006, M1-I1005, M1-I1004, M1-N1003, M1-V1002, M1-C1001, M1-Y1000,M1-I999, M1-V998, M1-R997, M1-G996, M1-D995, M1-S994, M1-R993, M1-F992,M1-P991, M1-Q990, M1-D989, M1-Q988, M1-L987, M1-R986, M1-L985, M1-I984,M1-M983, M1-G982, M1-V981, M1-S980, M1-F979, M1-L978, M1-L977, M1-I976,M1-A975, M1-I974, M1-L973, M1-D972, M1-T971, M1-V970, M1-N969, M1-W968,M1-Y967, M1-E966, M1-Q965, M1-L964, M1-W963, M1-V962, M1-K961, M1-V960,M1-K959, M1-Q958, M1-L957, M1-L956, M1-K955, M1-G954, M1-P953, M1-E952,M1-S951, M1-M950, M1-L949, M1-I948, M1-E947, M1-R946, M1-M945, M1-K944,M1-E943, M1-I942, M1-G941, M1-L940, M1-T939, M1-F938, M1-I937, M1-Y936,M1-S935, M1-I934, M1-V933, M1-I932, M1-W931, M1-E930, M1-Q929, M1-T928,M1-S927, M1-P926, M1-W925, M1-R924, M1-E923, M1-M922, M1-K921, M1-V920,M1-L919, M1-V918, M1-I917, M1-Y916, M1-N915, M1-F914, M1-L913, M1-M912,M1-L911, M1-Y910, M1-G909, M1-I908, M1-Y907, M1-A906, M1-L905, M1-T904,M1-Y903, M1-F902, M1-W901, M1-F900, M1-K899, M1-V898, M1-I897, M1-P896,M1-A895, M1-N894, M1-Y893, M1-F892, M1-E891, M1-Y890, M1-I889, M1-K888,M1-R887, M1-G886, M1-L885, M1-P884, M1-I883, M1-L882, M1-R881, M1-H880,M1-K879, M1-S878, M1-Q877, M1-V876, M1-E875, M1-E874, M1-E873, M1-D872,M1-K871, M1-K870, M1-R869, M1-S868, M1-S867, M1-E866, M1-G865, M1-N864,M1-N863, M1-R862, M1-G861, M1-L860, M1-M859, M1-A858, M1-T857, M1-L856,M1-E855, M1-M854, M1-D853, M1-E852, M1-E851, M1-E850, M1-K849, M1-E848,M1-K847, M1-T846, M1-P845, M1-K844, M1-E843, M1-P842, M1-E841, M1-E840,M1-A839, M1-E838, M1-K837, M1-E836, M1-Q835, M1-L834, M1-H833, M1-I832,M1-E831, M1-Q830, M1-A829, M1-Q828, M1-S827, M1-M826, M1-Y825, M1-P824,M1-M823, M1-D822, M1-D821, M1-K820, M1-N819, M1-K818, M1-F817, M1-E816,M1-L815, M1-S814, M1-L813, M1-I812, M1-S811, M1-P810, M1-P809, M1-L808,M1-L807, M1-I806, M1-G805, M1-L804, M1-I803, M1-V802, M1-K801, M1-L800,M1-G799, M1-S798, M1-N797, M1-K796, M1-R795, M1-M794, M1-R793, M1-L792,M1-R791, M1-G790, M1-M789, M1-W788, M1-M787, M1-D786, M1-T785, M1-L784,M1-L783, M1-M782, M1-Q781, M1-S780, M1-C779, M1-T778, M1-H777, M1-A776,M1-I775, M1-F774, M1-D773, M1-R772, M1-H771, M1-K770, M1-A769, M1-A768,M1-V767, M1-A766, M1-L765, M1-Q764, M1-L763, M1-C762, M1-T761, M1-A760,M1-N759, M1-S758, M1-W757, M1-N756, M1-K755, M1-L754, M1-E753, M1-Y752,M1-T751, M1-L750, M1-L749, M1-K748, M1-M747, M1-A746, M1-L745, M1-Q744,M1-E743, M1-D742, M1-Q741, M1-K740, M1-Y739, M1-S738, M1-Q737, M1-D736,M1-L735, M1-L734, M1-E733, M1-V732, M1-A731, M1-L730, M1-Q729, M1-G728,M1-F727, M1-D726, M1-R725, M1-S724, M1-N723, M1-H722, M1-N721, M1-L720,M1-E719, M1-Q718, M1-S717, M1-I716, M1-D715, M1-D714, M1-V713, M1-M712,M1-D711, M1-N710, M1-E709, M1-S708, M1-A707, M1-E706, M1-H705, M1-A704,M1-M703, M1-A702, M1-K701, M1-C700, M1-L699, M1-K698, M1-C697, M1-A696,M1-V695, M1-L694, M1-A693, M1-K692, M1-A691, M1-M690, M1-A689, M1-E688,M1-E687, M1-G686, M1-H685, M1-Q684, M1-W683, M1-F682, M1-F681, M1-L680,M1-A679, M1-M678, M1-K677, M1-Q676, M1-R675, M1-K674, M1-M673, M1-L672,M1-V671, M1-A670, M1-W669, M1-V668, M1-M667, M1-L666, M1-E665, M1-H664,M1-F663, M1-P662, M1-F661, M1-P660, M1-F659, M1-H658, M1-N657, M1-I656,M1-E655, M1-P654, M1-D653, M1-D652, M1-L651, M1-D650, M1-I649, M1-D648,M1-V647, M1-E646, M1-E645, M1-E644, M1-R643, M1-K642, M1-K641, M1-T640,M1-T639, M1-K638, M1-R637, M1-G636, M1-R635, M1-R634, M1-L633, M1-P632,M1-I631, M1-D630, M1-D629, M1-E628, M1-M627, M1-G626, M1-L625, M1-L624,M1-K623, M1-L622, M1-A621, M1-K620, M1-P619, M1-R618, M1-K617, M1-P616,M1-G615, M1-F614, M1-L613, M1-N612, M1-H611, M1-Y610, M1-L609, M1-T608,M1-R607, M1-F606, M1-R605, M1-K604, M1-R603, M1-T602, M1-Y601, M1-N600,M1-C599, M1-R598, M1-Y597, M1-A596, M1-G595, M1-G594, M1-M593, M1-L592,M1-Y591, M1-E590, M1-I589, M1-V588, M1-L587, M1-G586, M1-I585, M1-D584,M1-I583, M1-L582, M1-S581, M1-I580, M1-R579, M1-Y578, M1-D577, M1-P576,M1-P575, M1-L574, M1-N573, M1-G572, M1-K571, M1-K570, M1-V569, M1-D568,M1-R567, M1-V566, M1-L565, M1-H564, M1-Y563, M1-L562, M1-T561, M1-N560,M1-S559, M1-P558, M1-G557, M1-H556, M1-R555, M1-T554, M1-N553, M1-Y552,M1-L551, M1-E550, M1-E549, M1-L548, M1-R547, M1-S546, M1-I545, M1-T544,M1-L543, M1-F542, M1-R541, M1-H540, M1-M539, M1-S538, M1-V537, M1-G536,M1-N535, M1-E534, M1-I533, M1-L532, M1-L531, M1-K530, M1-V529, M1-F528,M1-D527, M1-V526, M1-R525, M1-D524, M1-L523, M1-V522, M1-L521, M1-A520,M1-D519, M1-L518, M1-M517, M1-A516, M1-Q515, M1-E514, M1-L513, M1-S512,M1-G511, M1-V510, M1-P509, M1-W508, M1-Q507, M1-Q506, M1-G505, M1-Y504,M1-I503, M1-F502, M1-I501, M1-Q500, M1-S499, M1-R498, M1-A497, M1-I496,M1-D495, M1-V494, M1-R493, M1-N492, M1-W491, M1-A490, M1-L489, M1-A488,M1-L487, M1-S486, M1-L485, M1-Q484, M1-D483, M1-P482, M1-A481, M1-S480,M1-A479, M1-N478, M1-A477, M1-G476, M1-K475, M1-L474, M1-L473, M1-A472,M1-T471, M1-L470, M1-I469, M1-A468, M1-L467, M1-D466, M1-I465, M1-D464,M1-Q463, M1-H462, M1-G461, M1-E460, M1-S459, M1-G458, M1-M457, M1-R456,M1-F455, M1-V454, M1-T453, M1-I452, M1-L451, M1-E450, M1-K449, M1-K448,M1-K447, M1-M446, M1-C445, M1-E444, M1-M443, M1-L442, M1-I441, M1-I440,M1-F439, M1-L438, M1-H437, M1-Q436, M1-A435, M1-Q434, M1-T433, M1-R432,M1-T431, M1-Y430, M1-T429, M1-F428, M1-T427, M1-K426, M1-Q425, M1-I424,M1-T423, M1-V422, M1-L421, M1-L420, M1-Q419, M1-D418, M1-R417, M1-L416,M1-S415, M1-E414, M1-N413, M1-I412, M1-L411, M1-G410, M1-G409, M1-E408,M1-E407, M1-S406, M1-Y405, M1-K404, M1-H403, M1-G402, M1-F401, M1-A400,M1-L399, M1-I398, M1-D397, M1-S396, M1-A395, M1-R394, M1-G393, M1-S392,M1-G391, M1-D390, M1-C389, M1-V388, M1-V387, M1-V386, M1-P385, M1-V384,M1-P383, M1-P382, M1-T381, M1-D380, M1-R379, M1-L378, M1-Y377, M1-E376,M1-L375, M1-V374, M1-I373, M1-S372, M1-I371, M1-V370, M1-N369, M1-P368,M1-G367, M1-G366, M1-E365, M1-V364, M1-I363, M1-L362, M1-A361, M1-V360,M1-V359, M1-P358, M1-V357, M1-G356, M1-Q355, M1-G354, M1-I353, M1-G352,M1-V351, M1-S350, M1-D349, M1-L348, M1-Q347, M1-C346, M1-S345, M1-G344,M1-W343, M1-F342, M1-S341, M1-Y340, M1-F339, M1-L338, M1-R337, M1-S336,M1-D335, M1-L334, M1-S333, M1-F332, M1-F331, M1-P330, M1-L329, M1-C328,M1-R327, M1-T326, M1-N325, M1-I324, M1-K323, M1-Q322, M1-L321, M1-S320,M1-I319, M1-H318, M1-K317, M1-E316, M1-L315, M1-Q314, M1-R313, M1-R312,M1-L311, M1-K310, M1-V309, M1-E308, M1-A307, M1-G306, M1-Y305, M1-K304,M1-G303, M1-T302, M1-T301, M1-G300, M1-N299, M1-D298, M1-A297, M1-L296,M1-I295, M1-F294, M1-H293, M1-S292, M1-H291, M1-M290, M1-S289, M1-N288,M1-L287, M1-V286, M1-T285, M1-L284, M1-K283, M1-S282, M1-M281, M1-P280,M1-N279, M1-S278, M1-M277, M1-T276, M1-Q275, M1-Y274, M1-P273, M1-R272,M1-V271, M1-V270, M1-D269, M1-R268, M1-G267, M1-I266, M1-L265, M1-D264,M1-E263, M1-Q262, M1-N261, M1-E260, M1-V259, M1-I258, M1-G257, M1-W256,M1-P255, M1-A254, M1-I253, M1-G252, M1-I251, M1-T250, M1-C249, M1-I248,M1-K247, M1-G246, M1-R245, M1-S244, M1-K243, M1-S242, M1-A241, M1-H240,M1-D239, M1-K238, M1-L237, M1-A236, M1-D235, M1-G234, M1-V233, M1-H232,M1-R231, M1-I230, M1-V229, M1-G228, M1-T227, M1-N226, M1-V225, M1-G224,M1-G223, M1-T222, M1-F221, M1-I220, M1-W219, M1-A218, M1-G217, M1-T216,M1-T215, M1-M214, M1-A213, M1-A212, M1-K211, M1-I210, M1-L209, M1-G208,M1-K207, M1-G206, M1-F205, M1-V204, M1-Q203, M1-K202, M1-L201, M1-K200,M1-P199, M1-Q198, M1-L197, M1-E196, M1-F195, M1-N194, M1-Q193, M1-L192,M1-G191, M1-G190, M1-H189, M1-V188, M1-S187, M1-I186, M1-L185, M1-L184,M1-K183, M1-P182, M1-L181, M1-E180, M1-L179, M1-Q178, M1-W177, M1-E176,M1-K175, M1-T174, M1-M173, M1-L172, M1-H171, M1-L170, M1-L169, M1-L168,M1-D167, M1-P166, M1-K165, M1-T164, M1-D163, M1-F162, M1-S161, M1-V160,M1-R159, M1-V158, M1-Y157, M1-M156, M1-A155, M1-K154, M1-N153, M1-S152,M1-H151, M1-G150, M1-G149, M1-G148, M1-Q147, M1-F146, M1-E145, M1-I144,M1-T143, M1-G142, M1-F141, M1-A140, M1-D139, M1-T138, M1-P137, M1-S136,M1-L135, M1-Q134, M1-T133, M1-H132, M1-K131, M1-S130, M1-I129, M1-S128,M1-W127, M1-K126, M1-E125, M1-S124, M1-Q123, M1-I122, M1-D121, M1-N120,M1-R119, M1-S118, M1-L117, M1-R116, M1-S115, M1-E114, M1-N113, M1-K112,M1-E11i, M1-N111, M1-Q109, M1-L108, M1-V107, M1-S106, M1-I105, M1-S104,M1-P103, M1-T102, M1-L101, M1-G100, M1-V99, M1-H98, M1-Q97, M1-G96,M1-I95, M1-L94, M1-R93, M1-G92, M1-C91, M1-C90, M1-C89, M1-R88, M1-H87,M1-P86, M1-D85, M1-K84, M1-T83, M1-S82, M1-P81, M1-I80, M1-I79, M1-H78,M1-V77, M1-C76, M1-E75, M1-R74, M1-K73, M1-Y72, M1-F71, M1-A70, M1-R69,M1-E68, M1-I67, M1-W66, M1-S65, M1-K64, M1-Q63, M1-A62, M1-P61, M1-T60,M1-Q59, M1-Q58, M1-S57, M1-P56, M1-L55, M1-G54, M1-S53, M1-R52, M1-K51,M1-T50, M1-S49, M1-G48, M1-Q47, M1-K46, M1-S45, M1-S44, M1-L43, M1-R42,M1-K41, M1-W40, M1-S39, M1-E38, M1-A37, M1-F36, M1-R35, M1-G34, M1-R33,M1-S32, M1-A31, M1-S30, M1-R29, M1-S28, M1-R27, M1-R26, M1-R25, M1-S24,M1-E23, M1-A22, M1-N21, M1-D20, M1-E19, M1-R18, M1-D17, M1-S16, M1-C15,M1-G14, M1-R13, M1-E12, M1-M11, M1-E10, M1-A9, M1-A8, and/or M1-D7 ofSEQ ID NO:12. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal LTRPC3l deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

[0589] Alternatively, preferred polypeptides of the present inventionmay comprise polypeptide sequences corresponding to, for example,internal regions of the LTRPC3l polypeptide (e.g., any combination ofboth N- and C-terminal LTRPC3l polypeptide deletions) of SEQ ID NO:12.For example, internal regions could be defined by the equation: aminoacid NX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of LTRPC3l (SEQ ID NO:12), and where CX refers toany C-terminal deletion polypeptide amino acid of LTRPC3l (SEQ IDNO:12). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

[0590] Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 5262 ofSEQ ID NO:11, b is an integer between 15 to 5276, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:11, and where b is greater than or equal to a+14. TABLE I ATCC NTTotal 5′ NT Deposit SEQ NT Seq of Start 3′ NT AA Total Gene CDNA No. Zand ID. of Codon of Seq ID AA of No. CloneID Date Vector No. X Clone ofORF ORF No. Y ORF 1. LTRPC3g N/A N/A 1 5201 72 5198 2 1709 (alsoreferred to as TRPM3g, and/or LTRPC6g) 2 LTRPC3h N/A N/A 3 5237 72 52344 1721 (also referred to as TRPM3h, and/or LTRPC6h) 3. LTRPC3I N/A N/A 55237 72 5234 6 1721 (also referred to as TRPM3I, and/or LTRPC6I) 4LTRPC3j N/A N/A 7 5171 72 5168 8 1699 (also referred to as TRPM3j,and/or LTRPC6j) 5 LTRPC3k N/A N/A 9 5207 72 5204 10 1711 (also referredto as TRPM3k, and/or LTRPC6k) 6 LTRPC3l N/A N/A 11 5276 72 5273 12 1734(also referred to as TRPM3l, and/or LTRPCl)

[0591] Table 1 summarizes the information corresponding to each “GeneNo.” described above. The nucleotide sequence identified as “NT SEQ IDNO:X” was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table 1 and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:X.

[0592] The cDNA Clone ID was deposited on the date and given thecorresponding deposit number listed in “ATCC Deposit No:Z and Date.”“Vector” refers to the type of vector contained in the cDNA Clone ID.

[0593] “Total NT Seq. Of Clone” refers to the total number ofnucleotides in the clone contig identified by “Gene No.” The depositedclone may contain all or most of the sequence of SEQ ID NO:X. Thenucleotide position of SEQ ID NO:X of the putative start codon(methionine) is identified as “5′ NT of Start Codon of ORF.”

[0594] The translated amino acid sequence, beginning with themethionine, is identified as “AA SEQ ID NO:Y,” although other readingframes can also be easily translated using known molecular biologytechniques. The polypeptides produced by these alternative open readingframes are specifically contemplated by the present invention.

[0595] The total number of amino acids within the open reading frame ofSEQ ID NO:Y is identified as “Total AA of ORF”.

[0596] SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:X is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:Xor the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:Y may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table 1.

[0597] Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

[0598] Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1, 3, 5, 7, 9 and/or 11 and the predicted translated amino acidsequence identified as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, but also asample of plasmid DNA containing a cDNA of the invention deposited withthe ATCC, as set forth in Table 1. The nucleotide sequence of eachdeposited clone can readily be determined by sequencing the depositedclone in accordance with known methods. The predicted amino acidsequence can then be verified from such deposits. Moreover, the aminoacid sequence of the protein encoded by a particular clone can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell containing the deposited cDNA, collecting theprotein, and determining its sequence.

[0599] The present invention also relates to the genes corresponding toSEQ ID NO:1, 3, 5, 7, 9 and/or 11, SEQ ID NO:2, 4, 6, 8, 10 and/or 12,or the deposited clone. The corresponding gene can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include preparing probes or primers from thedisclosed sequence and identifying or amplifying the corresponding genefrom appropriate sources of genomic material.

[0600] Also provided in the present invention are species homologs,allelic variants, and/or orthologs. The skilled artisan could, usingprocedures well-known in the art, obtain the polynucleotide sequencecorresponding to full-length genes (including, but not limited to thefull-length coding region), allelic variants, splice variants,orthologs, and/or species homologues of genes corresponding to SEQ IDNO:1, 3, 5, 7, 9 and/or 11, SEQ ID NO:2, 4, 6, 8, 10 and/or 12, or adeposited clone, relying on the sequence from the sequences disclosedherein or the clones deposited with the ATCC. For example, allelicvariants and/or species homologues may be isolated and identified bymaking suitable probes or primers which correspond to the 5′, 3′, orinternal regions of the sequences provided herein and screening asuitable nucleic acid source for allelic variants and/or the desiredhomologue.

[0601] The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0602] The polypeptides may be in the form of the protein, or may be apart of a larger protein, such as a fusion protein (see below). It isoften advantageous to include an additional amino acid sequence whichcontains secretory or leader sequences, pro-sequences, sequences whichaid in purification, such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0603] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

[0604] The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1, 3,5, 7, 9 and/or 11, and/or a cDNA provided in ATCC Deposit No. Z:. Thepresent invention also provides a polypeptide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:2, 4,6, 8, 10 and/or 12, and/or a polypeptide encoded by the cDNA provided inATCC Deposit No:PTA-4175. The present invention also providespolynucleotides encoding a polypeptide comprising, or alternativelyconsisting of the polypeptide sequence of SEQ ID NO:2, 4, 6, 8, 10and/or 12, and/or a polypeptide sequence encoded by the cDNA containedin ATCC Deposit No:PTA-4175.

[0605] Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1, 3, 5, 7, 9 and/or 11, and/or a cDNA provided in ATCCDeposit No.: that is less than, or equal to, a polynucleotide sequencethat is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 megabasepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs inlength.

[0606] The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1, 3, 5, 7, 9 and/or 11, the sequence containedin a deposit, and/or the nucleic acid sequence encoding the sequencedisclosed as SEQ ID NO:2, 4, 6, 8, 10 and/or 12.

[0607] The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystingent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table 2 below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R. TABLE II HyridizationStringency Polynucleotide Hybrid Length Temperature and Wash TemperatureCondition Hybrid± (bp)‡ Buffer† and Buffer† A DNA:DNA > or equal to 5065° C.; 1xSSC -or- 65° C.; 42° C.; 1xSSC, 0.3xSSC 50% formamide BDNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > or equal to 50 67° C.;1xSSC -or- 67° C.; 45° C.; 1xSSC, 0.3xSSC 50% formamide D DNA:RNA <50Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to 50 70° C.; 1xSSC -or- 70°C.; 50° C.; 1xSSC, 0.3xSSC 50% formamide F RNA:RNA <50 Tf*; 1xSSC Tf*;1xSSC G DNA:DNA > or equal to 50 65° C.; 4xSSC -or- 65° C.; 1xSSC 45°C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA >or equal to 50 67° C.; 4xSSC -or- 67° C.; 1xSSC 45° C.; 4xSSC, 50%formamide J DNA:RNA <50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA > or equal to 5070° C.; 4xSSC -or- 67° C.; 1xSSC 40° C.; 6xSSC, 50% formamide L RNA:RNA<50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA > or equal to 50 50° C.; 4xSSC -or-50° C.; 2xSSC 40° C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*; 6xSSC Tn*;6xSSC O DNA:RNA > or equal to 50 55° C.; 4xSSC -or- 55° C.; 2xSSC 42°C.; 6xSSC, 50% formamide P DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA >or equal to 50 60° C.; 4xSSC -or- 60° C.; 2xSSC 45° C.; 6xSSC, 50%formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC #and identifying theregion or regions of optimal sequence complementarity. Methods ofaligning two or more polynucleotide sequences and/or determining thepercent identity between two polynucleotide sequences are well known inthe art (e.g., MegAlign program of the DNA*Star suite of programs, etc).#hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 +16.6(log₁₀[Na+]) + 0.41 (% G + C) − (600/N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer ([NA+] for 1xSSC = .165 M).

[0608] Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

[0609] Preferably, such hybridizing polynucleotides have at least 70%sequence identity (more preferably, at least 80% identity; and mostpreferably at least 90% or 95% identity) with the polynucleotide of thepresent invention to which they hybridize, where sequence identity isdetermined by comparing the sequences of the hybridizing polynucleotideswhen aligned so as to maximize overlap and identity while minimizingsequence gaps. The determination of identity is well known in the art,and discussed more specifically elsewhere herein.

[0610] The invention encompasses the application of PCR methodology tothe polynucleotide sequences of the present invention, the clonedeposited with the ATCC, and/or the cDNA encoding the polypeptides ofthe present invention. PCR techniques for the amplification of nucleicacids are described in U.S. Pat. No. 4,683,195 and Saiki et al.,Science, 239:487-491 (1988). PCR, for example, may include the followingsteps, of denaturation of template nucleic acid (if double-stranded),annealing of primer to target, and polymerization. The nucleic acidprobed or used as a template in the amplification reaction may begenomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specificsequences from genomic DNA, specific RNA sequence, and/or cDNAtranscribed from mRNA. References for the general use of PCR techniques,including specific method parameters, include Mullis et al., Cold SpringHarbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology,Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650,(1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds.,Innis et al., Academic Press, New York, (1990).

Polynucleotide and Polypeptide Variants

[0611] The present invention also encompases variants (e.g., allelicvariants, orthologs, etc.) of the polynucleotide sequence disclosedherein in SEQ ID NO:1, 3, 5, 7, 9 and/or 11, the complementary strandthereto, and/or the cDNA sequence contained in the deposited clone.

[0612] The present invention also encompasses variants of thepolypeptide sequence, and/or fragments therein, disclosed in SEQ IDNO:2, 4, 6, 8, 10 and/or 12, a polypeptide encoded by the polunucleotidesequence in SEQ ID NO:1, 3, 5, 7, 9 and/or 11, and/or a polypeptideencoded by a cDNA in the deposited clone. “Variant” refers to apolynucleotide or polypeptide differing from the polynucleotide orpolypeptide of the present invention, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the polynucleotide or polypeptide of the presentinvention.

[0613] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising, or alternatively consisting of, apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding a LTRPC3 relatedpolypeptide having an amino acid sequence as shown in the sequencelisting and described in SEQ ID NO:1, 3, 5, 7, 9 and/or 11 or the cDNAcontained in ATCC Deposit No:PTA-4175; (b) a nucleotide sequenceencoding a mature LTRPC3 related polypeptide having the amino acidsequence as shown in the sequence listing and described in SEQ ID NO:1,3, 5, 7, 9 and/or 11 or the cDNA contained in ATCC Deposit No:PTA-4175;(c) a nucleotide sequence encoding a biologically active fragment of aLTRPC3 related polypeptide having an amino acid sequence shown in thesequence listing and described in SEQ ID NO:1, 3, 5, 7, 9 and/or 11 orthe cDNA contained in ATCC Deposit No:PTA-4175; (d) a nucleotidesequence encoding an antigenic fragment of a LTRPC3 related polypeptidehaving an amino acid sequence shown in the sequence listing anddescribed in SEQ ID NO:1, 3, 5, 7, 9 and/or 11 or the cDNA contained inATCC Deposit No:PTA-4175; (e) a nucleotide sequence encoding a LTRPC3related polypeptide comprising the complete amino acid sequence encodedby a human cDNA plasmid containined in SEQ ID NO:1, 3, 5, 7, 9 and/or 11or the cDNA contained in ATCC Deposit No:PTA-4175; (f) a nucleotidesequence encoding a mature LTRPC3 realted polypeptide having an aminoacid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1,3, 5, 7, 9 and/or 11 or the cDNA contained in ATCC Deposit No:PTA-4175;(g) a nucleotide sequence encoding a biologically active fragement of aLTRPC3 related polypeptide having an amino acid sequence encoded by ahuman cDNA plasmid contained in SEQ ID NO:1, 3, 5, 7, 9 and/or 11 or thecDNA contained in ATCC Deposit No:PTA-4175; (h) a nucleotide sequenceencoding an antigenic fragment of a LTRPC3 related polypeptide having anamino acid sequence encoded by a human cDNA plasmid contained in SEQ IDNO:1, 3, 5, 7, 9 and/or 11 or the cDNA contained in ATCC DepositNo:PTA-4175; (I) a nucleotide sequence complimentary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h),above.

[0614] The present invention is also directed to polynucleotidesequences which comprise, or alternatively consist of, a polynucleotidesequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to, for example, any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecule which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

[0615] Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a LTRPC3 related polypeptide having anamino acid sequence as shown in the sequence listing and described inTable 1; (b) a nucleotide sequence encoding a mature LTRPC3 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and described in Table 1; (c) a nucleotide sequence encoding abiologically active fragment of a LTRPC3 related polypeptide having anamino acid sequence as shown in the sequence listing and described inTable 1; (d) a nucleotide sequence encoding an antigenic fragment of aLTRPC3 related polypeptide having an amino acid sequence as shown in thesequence listing and described in Table 1; (e) a nucleotide sequenceencoding a LTRPC3 related polypeptide comprising the complete amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table 1; (f) a nucleotide sequence encoding amature LTRPC3 related polypeptide having an amino acid sequence encodedby a human cDNA in a cDNA plasmid contained in the ATCC Deposit anddescribed in Table 1: (g) a nucleotide sequence encoding a biologicallyactive fragment of a LTRPC3 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table 1; (h) a nucleotide sequence encoding anantigenic fragment of a LTRPC3 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCdeposit and described in Table 1; (i) a nucleotide sequencecomplimentary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h) above.

[0616] The present invention is also directed to nucleic acid moleculeswhich comprise, or alternatively, consist of, a nucleotide sequencewhich is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identical to, for example, any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h), above.

[0617] The present invention encompasses polypeptide sequences whichcomprise, or alternatively consist of, an amino acid sequence which isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, the following non-limited examples, the polypeptidesequence identified as SEQ ID NO:2, 4, 6, 8, 10 and/or 12, thepolypeptide sequence encoded by a cDNA provided in the deposited clone,and/or polypeptide fragments of any of the polypeptides provided herein.Polynucleotides encoded by these nucleic acid molecules are alsoencompassed by the invention. In another embodiment, the inventionencompasses nucleic acid molecule which comprise, or alternatively,consist of a polynucleotide which hybridizes under stringent conditions,or alternatively, under lower stringency conditions, to a polynucleotidein (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotideswhich hybridize to the complement of these nucleic acid molecules understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as arepolypeptides encoded by these polypeptides.

[0618] The present invention is also directed to polypeptides whichcomprise, or alternatively consist of, an amino acid sequence which isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for example, the polypeptide sequence shown in SEQ IDNO:2, 4, 6, 8, 10 and/or 12, a polypeptide sequence encoded by thenucleotide sequence in SEQ ID NO:1, 3, 5, 7, 9 and/or 11, a polypeptidesequence encoded by the cDNA in ATCC Deposit No:PTA-4175, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompasses by the present invention, asare the polypeptides encoded by these polynucleotides.

[0619] By a nucleic acid having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thenucleic acid is identical to the reference sequence except that thenucleotide sequence may include up to five point mutations per each 100nucleotides of the reference nucleotide sequence encoding thepolypeptide. In other words, to obtain a nucleic acid having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence referenced in Table 1, the ORF (open reading frame),or any fragment specified as described herein.

[0620] As a practical matter, whether any particular nucleic acidmolecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IkB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

[0621] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

[0622] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the CLUSTALW alignmentdoes not show a matched/alignment of the first 10 bases at 5′ end. The10 unpaired bases represent 10% of the sequence (number of bases at the5′ and 3′ ends not matched/total number of bases in the query sequence)so 10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

[0623] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a query amino acid sequence of the presentinvention, it is intended that the amino acid sequence of the subjectpolypeptide is identical to the query sequence except that the subjectpolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the query amino acid sequence. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a query amino acid sequence, up to 5% of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid. These alterations of the referencesequence may occur at the amino- or carboxy-terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0624] As a practical matter, whether any particular polypeptide is atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for instance, an amino acid sequence referenced in Table 1(SEQ ID NO:2) or to the amino acid sequence encoded by cDNA contained ina deposited clone, can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both amino acid sequences. The result ofsaid global sequence alignment is in percent identity. Preferredparameters used in a CLUSTALW alignment of DNA sequences to calculatepercent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1,Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, GapExtension Penalty=0.1, Scoring Method=Percent, Window Size=5 or thelength of the subject nucleotide sequence, whichever is shorter. Formultiple alignments, the following CLUSTALW parameters are preferred:Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap SeparationPenalty Range=8; End Gap Separation Penalty=Off; % Identity forAlignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic ResidueGap=Off; and Transition Weighting=0. The pairwise and multple alignmentparameters provided for CLUSTALW above represent the default parametersas provided with the AlignX software program (Vector NTI suite ofprograms, version 6.0).

[0625] The present invention encompasses the application of a manualcorrection to the percent identity results, in the instance where thesubject sequence is shorter than the query sequence because of N- orC-terminal deletions, not because of internal deletions. If only thelocal pairwise percent identity is required, no manual correction isneeded. However, a manual correction may be applied to determine theglobal percent identity from a global polypeptide alignment. Percentidentity calculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N- and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N- and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N- and C-terminal residuesof the subject sequence.

[0626] For example, a 90 amino acid residue subject sequence is alignedwith a 100 residue query sequence to determine percent identity. Thedeletion occurs at the N-terminus of the subject sequence and therefore,the CLUSTALW alignment does not show a matching/alignment of the first10 residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN- and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

[0627] In addition to the above method of aligning two or morepolynucleotide or polypeptide sequences to arrive at a percent identityvalue for the aligned sequences, it may be desirable in somecircumstances to use a modified version of the CLUSTALW algorithm whichtakes into account known structural features of the sequences to bealigned, such as for example, the SWISS-PROT designations for eachsequence. The result of such a modifed CLUSTALW algorithm may provide amore accurate value of the percent identity for two polynucleotide orpolypeptide sequences. Support for such a modified version of CLUSTALWis provided within the CLUSTALW algorithm and would be readilyappreciated to one of skill in the art of bioinformatics.

[0628] The variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.Polynucleotide variants can be produced for a variety of reasons, e.g.,to optimize codon expression for a particular host (change codons in themRNA to those preferred by a bacterial host such as E. coli).

[0629] Naturally occurring variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level and are included inthe present invention. Alternatively, non-naturally occurring variantsmay be produced by mutagenesis techniques or by direct synthesis.

[0630] Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

[0631] Moreover, ample evidence demonstrates that variants often retaina biological activity similar to that of the naturally occurringprotein. For example, Gayle and coworkers (J. Biol. Chem.268:22105-22111 (1993)) conducted extensive mutational analysis of humancytokine IL-1a. They used random mutagenesis to generate over 3,500individual IL-1a mutants that averaged 2.5 amino acid changes pervariant over the entire length of the molecule. Multiple mutations wereexamined at every possible amino acid position. The investigators foundthat “[m]ost of the molecule could be altered with little effect oneither [binding or biological activity].” In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

[0632] Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

[0633] Alternatively, such N-terminus or C-terminus deletions of apolypeptide of the present invention may, in fact, result in asignificant increase in one or more of the biological activities of thepolypeptide(s). For example, biological activity of many polypeptidesare governed by the presence of regulatory domains at either one or bothtermini. Such regulatory domains effectively inhibit the biologicalactivity of such polypeptides in lieu of an activation event (e.g.,binding to a cognate ligand or receptor, phosphorylation, proteolyticprocessing, etc.). Thus, by eliminating the regulatory domain of apolypeptide, the polypeptide may effectively be rendered biologicallyactive in the absence of an activation event.

[0634] The invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

[0635] The first strategy exploits the tolerance of amino acidsubstitutions by natural selection during the process of evolution. Bycomparing amino acid sequences in different species, conserved aminoacids can be identified. These conserved amino acids are likelyimportant for protein function. In contrast, the amino acid positionswhere substitutions have been tolerated by natural selection indicatesthat these positions are not critical for protein function. Thus,positions tolerating amino acid substitution could be modified whilestill maintaining biological activity of the protein.

[0636] The second strategy uses genetic engineering to introduce aminoacid changes at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

[0637] As the authors state, these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,most buried (within the tertiary structure of the protein) amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved.

[0638] The invention encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide of the presentinvention. Similarity is determined by conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a polypeptide by another amino acid of like characteristics(e.g., chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0639] Tolerated conservative amino acid substitutions of the presentinvention involve replacement of the aliphatic or hydrophobic aminoacids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Serand Thr; replacement of the acidic residues Asp and Glu; replacement ofthe amide residues Asn and Gln, replacement of the basic residues Lys,Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp,and replacement of the small-sized amino acids Ala, Ser, Thr, Met, andGly.

[0640] In addition, the present invention also encompasses theconservative substitutions provided in Table III below. TABLE III ForAmino Acid Code Replace with any of: Alanine A D-Ala, Gly, beta-Ala,L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met,Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu,D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln,D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine QD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, AcpIsoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile,D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa,His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4- carboxylic acid,D- or L-1- oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

[0641] Aside from the uses described above, such amino acidsubstitutions may also increase protein or peptide stability. Theinvention encompasses amino acid substitutions that contain, forexample, one or more non-peptide bonds (which replace the peptide bonds)in the protein or peptide sequence. Also included are substitutions thatinclude amino acid residues other than naturally occurring L-aminoacids, e.g., D-amino acids or non-naturally occurring or synthetic aminoacids, e.g., β or γ amino acids.

[0642] Both identity and similarity can be readily calculated byreference to the following publications: Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Informatics Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress,New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0643] In addition, the present invention also encompasses substitutionof amino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

[0644] Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

[0645] Besides conservative amino acid substitution, variants of thepresent invention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0646] For example, polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

[0647] Moreover, the invention further includes polypeptide variantscreated through the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:1, 3, 5, 7, 9and/or 11, the sequence of the clone submitted in a deposit, and/or thecDNA encoding the polypeptide disclosed as SEQ ID NO:2, 4, 6, 8, 10and/or 12. Such DNA Shuffling technology is known in the art and moreparticularly described elsewhere herein (e.g., WPC, Stemmer, PNAS,91:10747, (1994)), and in the Examples provided herein).

[0648] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of the present invention havingan amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of the present invention, which contains atleast one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of thepresent invention or fragments thereof (e.g., the mature form and/orother fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

[0649] The present invention is directed to polynucleotide fragments ofthe polynucleotides of the invention, in addition to polypeptidesencoded therein by said polynucleotides and/or fragments.

[0650] In the present invention, a “polynucleotide fragment” refers to ashort polynucleotide having a nucleic acid sequence which: is a portionof that contained in a deposited clone, or encoding the polypeptideencoded by the cDNA in a deposited clone; is a portion of that shown inSEQ ID NO:1, 3, 5, 7, 9 and/or 11 or the complementary strand thereto,or is a portion of a polynucleotide sequence encoding the polypeptide ofSEQ ID NO:2, 4, 6, 8, 10 and/or 12. The nucleotide fragments of theinvention are preferably at least about 15 nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt, at least about 50 nt, at leastabout 75 nt, or at least about 150 nt in length. A fragment “at least 20nt in length,” for example, is intended to include 20 or more contiguousbases from the cDNA sequence contained in a deposited clone or thenucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 9 and/or 11. In thiscontext “about” includes the particularly recited value, a value largeror smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus, or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

[0651] Moreover, representative examples of polynucleotide fragments ofthe invention, include, for example, fragments comprising, oralternatively consisting of, a sequence from about nucleotide number1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400,401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850,851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ IDNO:1, 3, 5, 7, 9 and/or 11, or the complementary strand thereto, or thecDNA contained in a deposited clone. In this context “about” includesthe particularly recited ranges, and ranges larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.Preferably, these fragments encode a polypeptide which has biologicalactivity. More preferably, these polynucleotides can be used as probesor primers as discussed herein. Also encompassed by the presentinvention are polynucleotides which hybridize to these nucleic acidmolecules under stringent hybridization conditions or lower stringencyconditions, as are the polypeptides encoded by these polynucleotides.

[0652] In the present invention, a “polypeptide fragment” refers to anamino acid sequence which is a portion of that contained in SEQ ID NO:2,4, 6, 8, 10 and/or 12 or encoded by the cDNA contained in a depositedclone. Protein (polypeptide) fragments may be “free-standing,” orcomprised within a larger polypeptide of which the fragment forms a partor region, most preferably as a single continuous region. Representativeexamples of polypeptide fragments of the invention, include, forexample, fragments comprising, or alternatively consisting of, fromabout amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120,121-140, 141-160, or 161 to the end of the coding region. Moreover,polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, or 150 amino acids in length. In this context“about” includes the particularly recited ranges or values, and rangesor values larger or smaller by several (5, 4, 3, 2, or 1) amino acids,at either extreme or at both extremes. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0653] Preferred polypeptide fragments include the full-length protein.Further preferred polypeptide fragments include the full-length proteinhaving a continuous series of deleted residues from the amino or thecarboxy terminus, or both. For example, any number of amino acids,ranging from 1-60, can be deleted from the amino terminus of thefull-length polypeptide. Similarly, any number of amino acids, rangingfrom 1-30, can be deleted from the carboxy terminus of the full-lengthprotein. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

[0654] Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2, 4, 6, 8, 10 and/or12 falling within conserved domains are specifically contemplated by thepresent invention. Moreover, polynucleotides encoding these domains arealso contemplated.

[0655] Other preferred polypeptide fragments are biologically activefragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thepolypeptide of the present invention. The biological activity of thefragments may include an improved desired activity, or a decreasedundesirable activity. Polynucleotides encoding these polypeptidefragments are also encompassed by the invention.

[0656] In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

[0657] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10 and/or 12, or an epitopeof the polypeptide sequence encoded by a polynucleotide sequencecontained in ATCC deposit No. Z or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1, 3, 5, 7, 9and/or 11 or contained in ATCC deposit No. Z under stringenthybridization conditions or lower stringency hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

[0658] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0659] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0660] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length, or longer. Additional non-exclusive preferredantigenic epitopes include the antigenic epitopes disclosed herein, aswell as portions thereof. Antigenic epitopes are useful, for example, toraise antibodies, including monoclonal antibodies, that specificallybind the epitope. Preferred antigenic epitopes include the antigenicepitopes disclosed herein, as well as any combination of two, three,four, five or more of these antigenic epitopes. Antigenic epitopes canbe used as the target molecules in immunoassays. (See, for instance,Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)).

[0661] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0662] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0663] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0664] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1, 3, 5, 7, 9 and/or 11 and the polypeptidesencoded by these polynucleotides may be achieved by DNA shuffling. DNAshuffling involves the assembly of two or more DNA segments byhomologous or site-specific recombination to generate variation in thepolynucleotide sequence. In another embodiment, polynucleotides of theinvention, or the encoded polypeptides, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of a polynucleotide encoding a polypeptide of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Antibodies

[0665] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, 4, 6, 8,10 and/or 12, and/or an epitope, of the present invention (as determinedby immunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, monovalent, bispecific,heteroconjugate, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) ismeant to include intact molecules, as well as, antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal or plant, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus,these fragments are preferred, as well as the products of a FAB or otherimmunoglobulin expression library. Moreover, antibodies of the presentinvention include chimeric, single chain, and humanized antibodies.

[0666] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0667] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0668] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0669] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homologue of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologuesof human proteins and the corresponding epitopes thereof. Antibodiesthat do not bind polypeptides with less than 95%, less than 90%, lessthan 85%, less than 80%, less than 75%, less than 70%, less than 65%,less than 60%, less than 55%, and less than 50% identity (as calculatedusing methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10−2 M, 10−2 M, 5×10−3 M, 10−3M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M, 10−6M, 5×10−7 M, 107 M,5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11 M, 10−11M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15M, or 10−15 M.

[0670] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0671] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0672] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 11(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman etal., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which areall incorporated by reference herein in their entireties).

[0673] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0674] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

[0675] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0676] The antibodies of the present invention may be generated by anysuitable method known in the art.

[0677] The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l protein is preparedand purified to render it substantially free of natural contaminants.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity. For example, apolypeptide of the invention can be administered to various host animalsincluding, but not limited to, rabbits, mice, rats, etc. to induce theproduction of sera containing polyclonal antibodies specific for theantigen. The administration of the polypeptides of the present inventionmay entail one or more injections of an immunizing agent and, ifdesired, an adjuvant. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art. For the purposesof the invention, “immunizing agent” may be defined as a polypeptide ofthe invention, including fragments, variants, and/or derivativesthereof, in addition to fusions with heterologous polypeptides and otherforms of the polypeptides described herein.

[0678] Typically, the immunizing agent and/or adjuvant will be injectedin the mammal by multiple subcutaneous or intraperitoneal injections,though they may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0679] The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

[0680] In a hybridoma method, a mouse, a humanized mouse, a mouse with ahuman immune system, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

[0681] The immunizing agent will typically include polypeptides of thepresent invention or a fusion protein thereof. Preferably, theimmunizing agent consists of an LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j,LTRPC3k, and/or LTRPC3l polypeptide or, more preferably, with a LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3lpolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986), pp. 59-103). Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0682] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. More preferred are the parent myeloma cellline (SP2O) as provided by the ATCC. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

[0683] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst the polypeptides of the present invention. Preferably, thebinding specificity of monoclonal antibodies produced by the hybridomacells is determined by immunoprecipitation or by an in vitro bindingassay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbantassay (ELISA). Such techniques are known in the art and within the skillof the artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

[0684] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods (Goding, supra, and/or according to Wands et al.(Gastroenterology 80:225-232 (1981)). Suitable culture media for thispurpose include, for example, Dulbecco's Modified Eagle's Medium andRPMI-1640. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0685] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-sepharose, hydroxyapatite chromatography, gelexclusion chromatography, gel electrophoresis, dialysis, or affinitychromatography.

[0686] The skilled artisan would acknowledge that a variety of methodsexist in the art for the production of monoclonal antibodies and thus,the invention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

[0687] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0688] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. Monoclonal antibodies can be prepared usinga wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

[0689] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

[0690] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0691] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0692] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0693] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

[0694] For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

[0695] In general, the humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

[0696] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety. The techniques of coleet al., and Boerder et al., are also available for the preparation ofhuman monoclonal antibodies (cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Riss, (1985); and Boemer et al., J. Immunol.,147(1):86-95, (1991)).

[0697] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

[0698] Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

[0699] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0700] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0701] Such anti-idiotypic antibodies capable of binding to the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and/or LTRPC3l polypeptide can beproduced in a two-step procedure. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody that binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones that produce an antibody whose ability to bind to theprotein-specific antibody can be blocked by the polypeptide. Suchantibodies comprise anti-idiotypic antibodies to the protein-specificantibody and can be used to immunize an animal to induce formation offurther protein-specific antibodies.

[0702] The antibodies of the present invention may be bispecificantibodies. Bispecific antibodies are monoclonal, Preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present invention, one of the bindingspecificities may be directed towards a polypeptide of the presentinvention, the other may be for any other antigen, and preferably for acell-surface protein, receptor, receptor subunit, tissue-specificantigen, virally derived protein, virally encoded envelope protein,bacterially derived protein, or bacterial surface protein, etc.

[0703] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0704] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

[0705] Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

[0706] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2, 4, 6, 8, 10 and/or 12.

[0707] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0708] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0709] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0710] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be. inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0711] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0712] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0713] More preferably, a clone encoding an antibody of the presentinvention may be obtained according to the method described in theExample section herein.

Methods of Producing Antibodies

[0714] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0715] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0716] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0717] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0718] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0719] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0720] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0721] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0722] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0723] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0724] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0725] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0726] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0727] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0728] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0729] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2, 4, 6, 8,10 and/or 12 may be fused or conjugated to the above antibody portionsto increase the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. Further, the polypeptidescorresponding to SEQ ID NO:2, 4, 6, 8, 10 and/or 12 may be fused orconjugated to the above antibody portions to facilitate purification.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4-polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide-linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

[0730] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0731] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

[0732] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

[0733] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0734] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0735] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0736] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0737] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0738] The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins' with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

[0739] During synthesis, the MIPs are imprinted so as to havecomplementary size, shape, charge and functional groups of the selectedtarget by using the target molecule itself (such as a polypeptide,antibody, etc.), or a substance having a very similar structure, as its“print” or “template.” MIPs can be derivatized with the same reagentsafforded to antibodies. For example, fluorescent “super” MIPs can becoated onto beads or wells for use in highly sensitive separations orassays, or for use in high throughput screening of proteins.

[0740] Moreover, MIPs based upon the structure of the polypeptide(s) ofthe present invention may be useful in screening for compounds that bindto the polypeptide(s) of the invention. Such a MIP would serve the roleof a synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

[0741] MIPs have also been shown to be useful in “sensing” the presenceof its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens,Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J.L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed usinga polypeptide of the present invention may be used in assays designed toidentify, and potentially quantitate, the level of said polypeptide in asample. Such a MIP may be used as a substitute for any componentdescribed in the assays, or kits, provided herein (e.g., ELISA, etc.).

[0742] A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

Uses for Antibodies Directed Against Polypeptides of the Invention

[0743] The antibodies of the present invention have various utilities.For example, such antibodies may be used in diagnostic assays to detectthe presence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

[0744] Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

[0745] Antibodies directed against the polypeptides of the presentinvention are useful for the affinity purification of such polypeptidesfrom recombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

Immunophenotyping

[0746] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0747] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

Assays for Antibody Binding

[0748] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0749] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0750] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0751] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0752] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., 3H or 125I) in the presence of increasingamounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

[0753] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0754] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0755] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0756] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0757] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10−2 M,10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, 10−5 M, 5×10−6 M,10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M,10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M,5×10−14 M, 10−14 M, 5×10−15 M, and 10−15 M.

[0758] Antibodies directed against polypeptides of the present inventionare useful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

[0759] Likewise, one could envision cloning the gene encoding anantibody directed against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

[0760] Alternatively, antibodies of the present invention could beproduced in a plant (e.g., cloning the gene of the antibody directedagainst a polypeptide of the present invention, and transforming a plantwith a suitable vector comprising said gene for constitutive expressionof the antibody within the plant), and the plant subsequently ingestedby an animal, thereby conferring temporary immunity to the animal forthe specific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

[0761] In another embodiment, antibodies of the present invention,preferably polyclonal antibodies, more preferably monoclonal antibodies,and most preferably single-chain antibodies, can be used as a means ofinhibiting gene expression of a particular gene, or genes, in a human,mammal, and/or other organism. See, for example, InternationalPublication Number WO 00/05391, published Feb. 3, 2000, to DowAgrosciences LLC. The application of such methods for the antibodies ofthe present invention are known in the art, and are more particularlydescribed elsewhere herein.

[0762] In yet another embodiment, antibodies of the present inventionmay be useful for multimerizing the polypeptides of the presentinvention. For example, certain proteins may confer enhanced biologicalactivity when present in a multimeric state (i.e., such enhancedactivity may be due to the increased effective concentration of suchproteins whereby more protein is available in a localized location).

Antibody-based Gene Therapy

[0763] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0764] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0765] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0766] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0767] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0768] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0769] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0770] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0771] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0772] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0773] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0774] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0775] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0776] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0777] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0778] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0779] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

[0780] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0781] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0782] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0783] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0784] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0785] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0786] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0787] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0788] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0789] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0790] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0791] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0792] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0793] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

Diagnosis and Imaging with Antibodies

[0794] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

[0795] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0796] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0797] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0798] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0799] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0800] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0801] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0802] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

[0803] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0804] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0805] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0806] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0807] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0808] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0809] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

[0810] Any polypeptide of the present invention can be used to generatefusion proteins. For example, the polypeptide of the present invention,when fused to a second protein, can be used as an antigenic tag.Antibodies raised against the polypeptide of the present invention canbe used to indirectly detect the second protein by binding to thepolypeptide. Moreover, because certain proteins target cellularlocations based on trafficking signals, the polypeptides of the presentinvention can be used as targeting molecules once fused to otherproteins.

[0811] Examples of domains that can be fused to polypeptides of thepresent invention include not only heterologous signal sequences, butalso other heterologous functional regions. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

[0812] Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

[0813] Moreover, polypeptides of the present invention, includingfragments, and specifically epitopes, can be combined with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), resulting in chimericpolypeptides. These fusion proteins facilitate purification and show anincreased half-life in vivo. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP A 394,827; Trauneckeret al., Nature 331:84-86 (1988).) Fusion proteins havingdisulfide-linked dimeric structures (due to the IgG) can also be moreefficient in binding and neutralizing other molecules, than themonomeric secreted protein or protein fragment alone. (Fountoulakis etal., J. Biochem. 270:3958-3964 (1995).)

[0814] Similarly, EP-A-0 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of the constantregion of immunoglobulin molecules together with another human proteinor part thereof. In many cases, the Fc part in a fusion protein isbeneficial in therapy and diagnosis, and thus can result in, forexample, improved pharmacokinetic properties. (EP-A 0232 262.)Alternatively, deleting the Fc part after the fusion protein has beenexpressed, detected, and purified, would be desired. For example, the Fcportion may hinder therapy and diagnosis if the fusion protein is usedas an antigen for immunizations. In drug discovery, for example, humanproteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995);K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

[0815] Moreover, the polypeptides of the present invention can be fusedto marker sequences (also referred to as “tags”). Due to theavailability of antibodies specific to such “tags”, purification of thefused polypeptide of the invention, and/or its identification issignificantly facilitated since antibodies specific to the polypeptidesof the invention are not required. Such purification may be in the formof an affinity purification whereby an anti-tag antibody or another typeof affinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

[0816] The skilled artisan would acknowledge the existence of other“tags” which could be readily substituted for the tags referred to suprafor purification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ IDNO:16), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

[0817] The present invention also encompasses the attachment of up tonine codons encoding a repeating series of up to nine arginine aminoacids to the coding region of a polynucleotide of the present invention.The invention also encompasses chemically derivitizing a polypeptide ofthe present invention with a repeating series of up to nine arginineamino acids. Such a tag, when attached to a polypeptide, has recentlybeen shown to serve as a universal pass, allowing compounds access tothe interior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

[0818] Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

[0819] Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N. Y. Acad. Sci. 1999;886:233-5), or HCtoxin (Tonukari N J, et al., Plant Cell. 2000 February;12(2):237-248),for example. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

[0820] The invention encompasses the fusion of antibodies directedagainst polypeptides of the present invention, including variants andfragments thereof, to said toxins for delivering the toxin to specificlocations in a cell, to specific tissues, and/or to specific species.Such bifunctional antibodies are known in the art, though a reviewdescribing additional advantageous fusions, including citations formethods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

[0821] Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

[0822] The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0823] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0824] The polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0825] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

[0826] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

[0827] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

[0828] A polypeptide of this invention can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

[0829] Polypeptides of the present invention, and preferably thesecreted form, can also be recovered from: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect, and mammalian cells. Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

[0830] In one embodiment, the yeast Pichia pastoris is used to expressthe polypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O2. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O2. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

[0831] In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

[0832] Many other yeast vectors could be used in place of pPIC9K, suchas, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-SI, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

[0833] In another embodiment, high-level expression of a heterologouscoding sequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

[0834] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., coding sequence), and/or toinclude genetic material (e.g., heterologous polynucleotide sequences)that is operably associated with the polynucleotides of the invention,and which activates, alters, and/or amplifies endogenouspolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous polynucleotide sequences via homologousrecombination, resulting in the formation of a new transcription unit(see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.5,733,761, issued Mar. 31, 1998; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0835] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example,a polypeptide corresponding to a fragment of a polypeptide sequence ofthe invention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0836] The invention encompasses polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0837] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

[0838] Also provided by the invention are chemically modifiedderivatives of the polypeptides of the invention which may provideadditional advantages such as increased solubility, stability andcirculating time of the polypeptide, or decreased immunogenicity (seeU.S. Pat. No. 4,179,337). The chemical moieties for derivitization maybe selected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The polypeptides may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

[0839] The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

[0840] The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

[0841] For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0842] Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

[0843] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0844] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0845] As with the various polymers exemplified above, it iscontemplated that the polymeric residues may contain functional groupsin addition, for example, to those typically involved in linking thepolymeric residues to the polypeptides of the present invention. Suchfunctionalities include, for example, carboxyl, amine, hydroxy and thiolgroups. These functional groups on the polymeric residues can be furtherreacted, if desired, with materials that are generally reactive withsuch functional groups and which can assist in targeting specifictissues in the body including, for example, diseased tissue. Exemplarymaterials which can be reacted with the additional functional groupsinclude, for example, proteins, including antibodies, carbohydrates,peptides, glycopeptides, glycolipids, lectins, and nucleosides.

[0846] In addition to residues of hydrophilic polymers, the chemicalused to derivatize the polypeptides of the present invention can be asaccharide residue. Exemplary saccharides which can be derived include,for example, monosaccharides or sugar alcohols, such as erythrose,threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitoland sedoheptulose, with preferred monosaccharides being fructose,mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides,such as lactose, sucrose, maltose and cellobiose. Other saccharidesinclude, for example, inositol and ganglioside head groups. Othersuitable saccharides, in addition to those exemplified above, will bereadily apparent to one skilled in the art based on the presentdisclosure. Generally, saccharides which may be used for derivitizationinclude saccharides that can be attached to the polypeptides of theinvention via alkylation or acylation reactions.

[0847] Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

[0848] The invention encompasses derivitization of the polypeptides ofthe present invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, argi nine, histidine, glucuronicacid, gluconic acid, glucaric acid, gal acturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof Accordingly, suitable polymers include, for example,proteins, such as albumin, polyalginates, and polylactide-coglycolidepolymers. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

[0849] Moreover, the invention encompasses additional modifications ofthe polypeptides of the present invention. Such additional modificationsare known in the art, and are specifically provided, in addition tomethods of derivitization, etc., in U.S. Pat. No. 6,028,066, which ishereby incorporated in its entirety herein.

[0850] The polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of thepolypeptides of the invention, their preparation, and compositions(preferably, Therapeutics) containing them. In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0851] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only polypeptides corresponding to the amino acid sequence ofSEQ ID NO:2, 4, 6, 8, 10 and/or 12 or encoded by the cDNA contained in adeposited clone (including fragments, variants, splice variants, andfusion proteins, corresponding to these polypeptides as describedherein). These homomers may contain polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only polypeptides having anidentical amino acid sequence. In another specific embodiment, a homomerof the invention is a multimer containing polypeptides having differentamino acid sequences. In specific embodiments, the multimer of theinvention is a homodimer (e.g., containing polypeptides having identicalor different amino acid sequences) or a homotrimer (e.g., containingpolypeptides having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

[0852] As used herein, the term heteromer refers to a multimercontaining one or more heterologous polypeptides (i.e., polypeptides ofdifferent proteins) in addition to the polypeptides of the invention. Ina specific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

[0853] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

[0854] In one example, covalent associations are between theheterologous sequence contained in a fusion protein of the invention(see, e.g., U.S. Pat. No. 5,478,925). In a specific example, thecovalent associations are between the heterologous sequence contained inan Fc fusion protein of the invention (as described herein). In anotherspecific example, covalent associations of fusion proteins of theinvention are between heterologous polypeptide sequence from anotherprotein that is capable of forming covalently associated multimers, suchas for example, osteoprotegerin (see, e.g., International PublicationNO: WO 98/49305, the contents of which are herein incorporated byreference in its entirety). In another embodiment, two or morepolypeptides of the invention are joined through peptide linkers.Examples include those peptide linkers described in U.S. Pat. No.5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple polypeptides of the invention separated by peptide linkers maybe produced using conventional recombinant DNA technology.

[0855] Another method for preparing multimer polypeptides of theinvention involves use of polypeptides of the invention fused to aleucine zipper or isoleucine zipper polypeptide sequence. Leucine zipperand isoleucine zipper domains are polypeptides that promotemultimerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, (1988)), and have since been found in avariety of different proteins. Among the known leucine zippers arenaturally occurring peptides and derivatives thereof that dimerize ortrimerize. Examples of leucine zipper domains suitable for producingsoluble multimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art.

[0856] Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

[0857] In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

[0858] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

[0859] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,polypeptides contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No. 5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain (orhydrophobic or signal peptide) and which can be incorporated by membranereconstitution techniques into liposomes (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).

[0860] In addition, the polynucleotide insert of the present inventioncould be operatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

[0861] Each of the polynucleotides identified herein can be used innumerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0862] The polynucleotides of the present invention are useful forchromosome identification. There exists an ongoing need to identify newchromosome markers, since few chromosome marking reagents, based onactual sequence data (repeat polymorphisms), are presently available.Each polynucleotide of the present invention can be used as a chromosomemarker.

[0863] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1,3, 5, 7, 9 and/or 11. Primers can be selected using computer analysis sothat primers do not span more than one predicted exon in the genomicDNA. These primers are then used for PCR screening of somatic cellhybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the SEQ ID NO:1, 3, 5, 7, 9and/or 11 will yield an amplified fragment.

[0864] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

[0865] Precise chromosomal location of the polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

[0866] For chromosome mapping, the polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0867] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

[0868] Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

[0869] Furthermore, increased or decreased expression of the gene inaffected organisms as compared to unaffected organisms can be assessedusing polynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

[0870] Thus, the invention also provides a diagnostic method usefulduring diagnosis of a disorder, involving measuring the expression levelof polynucleotides of the present invention in cells or body fluid froman organism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

[0871] By “measuring the expression level of a polynucleotide of thepresent invention” is intended qualitatively or quantitatively measuringor estimating the level of the polypeptide of the present invention orthe level of the mRNA encoding the polypeptide in a first biologicalsample either directly (e.g., by determining or estimating absoluteprotein level or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0872] By “biological sample” is intended any biological sample obtainedfrom an organism, body fluids, cell line, tissue culture, or othersource which contains the polypeptide of the present invention or mRNA.As indicated, biological samples include body fluids (such as thefollowing non-limiting examples, sputum, amniotic fluid, urine, saliva,breast milk, secretions, interstitial fluid, blood, serum, spinal fluid,etc.) which contain the polypeptide of the present invention, and othertissue sources found to express the polypeptide of the presentinvention. Methods for obtaining tissue biopsies and body fluids fromorganisms are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

[0873] The method(s) provided above may Preferably be applied in adiagnostic method and/or kits in which polynucleotides and/orpolypeptides are attached to a solid support. In one exemplary method,the support may be a “gene chip” or a “biological chip” as described inU.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a genechip with polynucleotides of the present invention attached may be usedto identify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The U.S. Patents referencedsupra are hereby incorporated by reference in their entirety herein.

[0874] The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

[0875] In addition to the foregoing, a polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Antisense techniques are discussed, for example, in Okano, J.Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance Lee et al., NucleicAcids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);and Dervan et al., Science 251: 1360 (1991). Both methods rely onbinding of the polynucleotide to a complementary DNA or RNA. For thesetechniques, preferred polynucleotides are usually oligonucleotides 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

[0876] The present invention encompasses the addition of a nuclearlocalization signal, operably linked to the 5′ end, 3′ end, or anylocation therein, to any of the oligonucleotides, antisenseoligonucleotides, triple helix oligonucleotides, ribozymes, PNAoligonucleotides, and/or polynucleotides, of the present invention. See,for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000);which is hereby incorporated herein by reference.

[0877] Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/GNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

[0878] The polynucleotides are also useful for identifying organismsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The polynucleotides of the presentinvention can be used as additional DNA markers for RFLP.

[0879] The polynucleotides of the present invention can also be used asan alternative to RFLP, by determining the actual base-by-base DNAsequence of selected portions of an organisms genome. These sequencescan be used to prepare PCR primers for amplifying and isolating suchselected DNA, which can then be sequenced. Using this technique,organisms can be identified because each organism will have a unique setof DNA sequences. Once an unique ID database is established for anorganism, positive identification of that organism, living or dead, canbe made from extremely small tissue samples. Similarly, polynucleotidesof the present invention can be used as polymorphic markers, in additionto, the identification of transformed or non-transformed cells and/ortissues.

[0880] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination. Moreover, as mentioned above, such reagents can beused to screen and/or identify transformed and non-transformed cellsand/or tissues.

[0881] In the very least, the polynucleotides of the present inventioncan be used as molecular weight markers on Southern gels, as diagnosticprobes for the presence of a specific mRNA in a particular cell type, asa probe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

[0882] Each of the polypeptides identified herein can be used innumerous ways. The following description should be considered exemplaryand utilizes known techniques.

[0883] A polypeptide of the present invention can be used to assayprotein levels in a biological sample using antibody-based techniques.For example, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0884] In addition to assaying protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0885] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, 131I, 112In, 99mTc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “lmmunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0886] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of a polypeptide of thepresent invention in cells or body fluid of an individual; (b) comparingthe level of gene expression with a standard gene expression level,whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof a disorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0887] Moreover, polypeptides of the present invention can be used totreat, prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

[0888] Similarly, antibodies directed to a polypeptide of the presentinvention can also be used to treat, prevent, and/or diagnose disease.For example, administration of an antibody directed to a polypeptide ofthe present invention can bind and reduce overproduction of thepolypeptide. Similarly, administration of an antibody can activate thepolypeptide, such as by binding to a polypeptide bound to a membrane(receptor).

[0889] At the very least, the polypeptides of the present invention canbe used as molecular weight markers on SDS-PAGE gels or on molecularsieve gel filtration columns using methods well known to those of skillin the art. Polypeptides can also be used to raise antibodies, which inturn are used to measure protein expression from a recombinant cell, asa way of assessing transformation of the host cell. Moreover, thepolypeptides of the present invention can be used to test the followingbiological activities.

Gene Therapy Methods

[0890] Another aspect of the present invention is to gene therapymethods for treating or preventing disorders, diseases and conditions.The gene therapy methods relate to the introduction of nucleic acid(DNA, RNA and antisense DNA or RNA) sequences into an animal to achieveexpression of a polypeptide of the present invention. This methodrequires a polynucleotide which codes for a polypeptide of the inventionthat operatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

[0891] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

[0892] As discussed in more detail below, the polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). Thepolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0893] In one embodiment, the polynucleotide of the invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotides of the invention can also be delivered in liposomeformulations and lipofectin formulations and the like can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

[0894] The polynucleotide vector constructs of the invention used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0895] Any strong promoter known to those skilled in the art can be usedfor driving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

[0896] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0897] The polynucleotide construct of the invention can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0898] For the naked nucleic acid sequence injection, an effectivedosage amount of DNA or RNA will be in the range of from about 0.05mg/kg body weight to about 50 mg/kg body weight. Preferably the dosagewill be from about 0.005 mg/kg to about 20 mg/kg and more preferablyfrom about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan ofordinary skill will appreciate, this dosage will vary according to thetissue site of injection. The appropriate and effective dosage ofnucleic acid sequence can readily be determined by those of ordinaryskill in the art and may depend on the condition being treated and theroute of administration.

[0899] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0900] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0901] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0902] In certain embodiments, the polynucleotide constructs of theinvention are complexed in a liposome preparation. Liposomalpreparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner et al.,Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA, 86:6077-6081 (1989), which is herein incorporated by reference);and purified transcription factors (Debs et al., J. Biol. Chem.,265:10189-10192 (1990), which is herein incorporated by reference), infunctional form.

[0903] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0904] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Feigner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0905] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0906] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0907] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

[0908] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0909] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication NO: WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0910] In certain embodiments, cells are engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding polypeptides of the invention. Retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

[0911] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy , 1:5-14 (1990), which is incorporated herein by reference inits entirety. The vector may transduce the packaging cells through anymeans known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO4 precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0912] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding polypeptides of theinvention. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express polypeptides of the invention.

[0913] In certain other embodiments, cells are engineered, ex vivo or invivo, with polynucleotides of the invention contained in an adenovirusvector. Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

[0914] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell , 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0915] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

[0916] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0917] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

[0918] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

[0919] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the desired endogenous polynucleotidesequence so the promoter will be operably linked to the endogenoussequence upon homologous recombination.

[0920] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0921] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0922] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

[0923] The polynucleotides encoding polypeptides of the presentinvention may be administered along with other polynucleotides encodingangiogenic proteins. Angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C),VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[0924] Preferably, the polynucleotide encoding a polypeptide of theinvention contains a secretory signal sequence that facilitatessecretion of the protein. Typically, the signal sequence is positionedin the coding region of the polynucleotide to be expressed towards or atthe 5′ end of the coding region. The signal sequence may be homologousor heterologous to the polynucleotide of interest and may be homologousor heterologous to the cells to be transfected. Additionally, the signalsequence may be chemically synthesized using methods known in the art.

[0925] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers. (Kaneda et al., Science, 243:375 (1989)).

[0926] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0927] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0928] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0929] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0930] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities

[0931] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

[0932] The polynucleotides or polypeptides, or agonists or antagonistsof the present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

[0933] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

[0934] Moreover, a polynucleotides or polypeptides, or agonists orantagonists of the present invention could also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could be used to treat or prevent bloodcoagulation diseases, disorders, and/or conditions (e.g.,afibrinogenemia, factor deficiencies), blood platelet diseases,disorders, and/or conditions (e.g. thrombocytopenia), or woundsresulting from trauma, surgery, or other causes. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that can decrease hemostatic or thrombolytic activitycould be used to inhibit or dissolve clotting. These molecules could beimportant in the treatment or prevention of heart attacks (infarction),strokes, or scarring.

[0935] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

[0936] Examples of autoimmune diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed or detected by thepresent invention include, but are not limited to: Addison's Disease,hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

[0937] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

[0938] A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD). Organrejection occurs by host immune cell destruction of the transplantedtissue through an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

[0939] Similarly, a polynucleotides or polypeptides, or agonists orantagonists of the present invention may also be used to modulateinflammation. For example, the polypeptide or polynucleotide or agonistsor antagonist may inhibit the proliferation and differentiation of cellsinvolved in an inflammatory response. These molecules can be used totreat, prevent, and/or diagnose inflammatory conditions, both chronicand acute conditions, including chronic prostatitis, granulomatousprostatitis and malacoplakia, inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

[0940] A polynucleotides or polypeptides, or agonists or antagonists ofthe invention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

[0941] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

[0942] Examples of hyperproliferative diseases, disorders, and/orconditions that can be treated, prevented, and/or diagnosed bypolynucleotides or. polypeptides, or agonists or antagonists of thepresent invention include, but are not limited to neoplasms located inthe: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

[0943] Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

[0944] One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

[0945] Thus, the present invention provides a method for treating orpreventing cell proliferative diseases, disorders, and/or conditions byinserting into an abnormally proliferating cell a polynucleotide of thepresent invention, wherein said polynucleotide represses saidexpression.

[0946] Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

[0947] Polynucleotides of the present invention may be useful inrepressing expression of oncogenic genes or antigens. By “repressingexpression of the oncogenic genes” is intended the suppression of thetranscription of the gene, the degradation of the gene transcript(pre-message RNA), the inhibition of splicing, the destruction of themessenger RNA, the prevention of the post-translational modifications ofthe protein, the destruction of the protein, or the inhibition of thenormal function of the protein.

[0948] For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

[0949] The polynucleotides of the present invention may be delivereddirectly to cell proliferative disorder/disease sites in internalorgans, body cavities and the like by use of imaging devices used toguide an injecting needle directly to the disease site. Thepolynucleotides of the present invention may also be administered todisease sites at the time of surgical intervention.

[0950] By “cell proliferative disease” is meant any human or animaldisease or disorder, affecting any one or any combination of organs,cavities, or body parts, which is characterized by single or multiplelocal abnormal proliferations of cells, groups of cells, or tissues,whether benign or malignant.

[0951] Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

[0952] The present invention is further directed to antibody-basedtherapies which involve administering of anti-polypeptides andanti-polynucleotide antibodies to a mammalian, preferably human, patientfor treating, preventing, and/or diagnosing one or more of the describeddiseases, disorders, and/or conditions. Methods for producinganti-polypeptides and anti-polynucleotide antibodies polyclonal andmonoclonal antibodies are described in detail elsewhere herein. Suchantibodies may be provided in pharmaceutically acceptable compositionsas known in the art or as described herein.

[0953] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0954] In particular, the antibodies, fragments and derivatives of thepresent invention are useful for treating, preventing, and/or diagnosinga subject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

[0955] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

[0956] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of diseases, disorders,and/or conditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10−6M,10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M,5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M,5×10−15M, and 10−15M.

[0957] Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

[0958] Polypeptides, including protein fusions, of the presentinvention, or fragments thereof may be useful in inhibitingproliferative cells or tissues through the induction of apoptosis. Saidpolypeptides may act either directly, or indirectly to induce apoptosisof proliferative cells and tissues, for example in the activation of adeath-domain receptor, such as tumor necrosis factor (TNF) receptor-1,CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein(TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and-2 (See Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998),which is hereby incorporated by reference). Moreover, in anotherpreferred embodiment of the present invention, said polypeptides mayinduce apoptosis through other mechanisms, such as in the activation ofother proteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses. 50(5):423-33 (1998), Chem. Biol. Interact. Apr.24;111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

[0959] Polypeptides, including protein fusions to, or fragments thereof,of the present invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

[0960] In another embodiment, the invention provides a method ofdelivering compositions containing the polypeptides of the invention(e.g., compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

[0961] Polypeptides, protein fusions to, or fragments thereof, of thepresent invention are useful in enhancing the immunogenicity and/orantigenicity of proliferating cells or tissues, either directly, such aswould occur if the polypeptides of the present invention ‘vaccinated’the immune response to respond to proliferative antigens and immunogens,or indirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

[0962] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention may be used to treat, prevent, and/or diagnosecardiovascular diseases, disorders, and/or conditions, includingperipheral artery disease, such as limb ischemia.

[0963] Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cortriatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

[0964] Cardiovascular diseases, disorders, and/or conditions alsoinclude heart disease, such as arrhythmias, carcinoid heart disease,high cardiac output, low cardiac output, cardiac tamponade, endocarditis(including bacterial), heart aneurysm, cardiac arrest, congestive heartfailure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema,heart hypertrophy, congestive cardiomyopathy, left ventricularhypertrophy, right ventricular hypertrophy, post-infarction heartrupture, ventricular septal rupture, heart valve diseases, myocardialdiseases, myocardial ischemia, pericardial effusion, pericarditis(including constrictive and tuberculous), pneumopericardium,postpericardiotomy syndrome, pulmonary heart disease, rheumatic heartdisease, ventricular dysfunction, hyperemia, cardiovascular pregnancycomplications, Scimitar Syndrome, cardiovascular syphilis, andcardiovascular tuberculosis.

[0965] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0966] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0967] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0968] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0969] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

[0970] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0971] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0972] Cerebrovascular diseases, disorders, and/or conditions includecarotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

[0973] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0974] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0975] Polynucleotides or polypeptides, or agonists or antagonists ofthe invention, are especially effective for the treatment of criticallimb ischemia and coronary disease.

[0976] Polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. Polypeptides of the invention may be administered aspart of a Therapeutic, described in more detail below. Methods ofdelivering polynucleotides of the invention are described in more detailherein.

Anti-Angiogenesis Activity

[0977] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye diseases, disorders, and/orconditions, and psoriasis. See, e.g., reviews by Moses et al., Biotech.9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763(1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman,Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press,New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982);and Folkman et al., Science 221:719-725 (1983). In a number ofpathological conditions, the process of angiogenesis contributes to thedisease state. For example, significant data have accumulated whichsuggest that the growth of solid tumors is dependent on angiogenesis.Folkman and Klagsbrun, Science 235:442-447 (1987).

[0978] The present invention provides for treatment of diseases,disorders, and/or conditions associated with neovascularization byadministration of the polynucleotides and/or polypeptides of theinvention, as well as agonists or antagonists of the present invention.Malignant and metastatic conditions which can be treated with thepolynucleotides and polypeptides, or agonists or antagonists of theinvention include, but are not limited to, malignancies, solid tumors,and cancers described herein and otherwise known in the art (for areview of such disorders, see Fishman et al., Medicine, 2d Ed., J. B.Lippincott Co., Philadelphia (1985)).Thus, the present inventionprovides a method of treating, preventing, and/or diagnosing anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat or prevent a cancer or tumor. Cancers which may betreated, prevented, and/or diagnosed with polynucleotides, polypeptides,antagonists and/or agonists include, but are not limited to solidtumors, including prostate, lung, breast, ovarian, stomach, pancreas,larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum,cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primarytumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma;leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advancedmalignancies; and blood born tumors such as leukemias. For example,polynucleotides, polypeptides, antagonists and/or agonists may bedelivered topically, in order to treat or prevent cancers such as skincancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

[0979] Within yet other aspects, polynucleotides, polypeptides,antagonists and/or agonists may be utilized to treat superficial formsof bladder cancer by, for example, intravesical administration.Polynucleotides, polypeptides, antagonists and/or agonists may bedelivered directly into the tumor, or near the tumor site, via injectionor a catheter. Of course, as the artisan of ordinary skill willappreciate, the appropriate mode of administration will vary accordingto the cancer to be treated. Other modes of delivery are discussedherein.

[0980] Polynucleotides, polypeptides, antagonists and/or agonists may beuseful in treating, preventing, and/or diagnosing other diseases,disorders, and/or conditions, besides cancers, which involveangiogenesis. These diseases, disorders, and/or conditions include, butare not limited to: benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas;artheroscleric plaques; ocular angiogenic diseases, for example,diabetic retinopathy, retinopathy of prematurity, macular degeneration,corneal graft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vesselgrowth) of the eye; rheumatoid arthritis; psoriasis; delayed woundhealing; endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

[0981] For example, within one aspect of the present invention methodsare provided for treating, preventing, and/or diagnosing hypertrophicscars and keloids, comprising the step of administering apolynucleotide, polypeptide, antagonist and/or agonist of the inventionto a hypertrophic scar or keloid.

[0982] Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., burns), and is preferablyinitiated after the proliferative phase has had time to progress(approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating, preventing, and/ordiagnosing neovascular diseases of the eye, including for example,corneal neovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

[0983] Moreover, Ocular diseases, disorders, and/or conditionsassociated with neovascularization which can be treated, prevented,and/or diagnosed with the polynucleotides and polypeptides of thepresent invention (including agonists and/or antagonists) include, butare not limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

[0984] Thus, within one aspect of the present invention methods areprovided for treating or preventing neovascular diseases of the eye suchas corneal neovascularization (including corneal graftneovascularization), comprising the step of administering to a patient atherapeutically effective amount of a compound (as described above) tothe cornea, such that the formation of blood vessels is inhibited.Briefly, the cornea is a tissue which normally lacks blood vessels. Incertain pathological conditions however, capillaries may extend into thecornea from the pericorneal vascular plexus of the limbus. When thecornea becomes vascularized, it also becomes clouded, resulting in adecline in the patient's visual acuity. Visual loss may become completeif the cornea completely opacitates. A wide variety of diseases,disorders, and/or conditions can result in corneal neovascularization,including for example, corneal infections (e.g., trachoma, herpessimplex keratitis, leishmaniasis and onchocerciasis), immunologicalprocesses (e.g., graft rejection and Stevens-Johnson's syndrome), alkaliburns, trauma, inflammation (of any cause), toxic and nutritionaldeficiency states, and as a complication of wearing contact lenses.

[0985] Within particularly preferred embodiments of the invention, maybe prepared for topical administration in saline (combined with any ofthe preservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

[0986] Within other embodiments, the compounds described above may beinjected directly into the corneal stroma by an ophthalmologist undermicroscopic guidance. The preferred site of injection may vary with themorphology of the individual lesion, but the goal of the administrationwould be to place the composition at the advancing front of thevasculature (i.e., interspersed between the blood vessels and the normalcornea). In most cases this would involve perilimbic corneal injectionto “protect” the cornea from the advancing blood vessels. This methodmay also be utilized shortly after a corneal insult in order toprophylactically prevent corneal neovascularization. In this situationthe material could be injected in the perilimbic cornea interspersedbetween the corneal lesion and its undesired potential limbic bloodsupply. Such methods may also be utilized in a similar fashion toprevent capillary invasion of transplanted corneas. In asustained-release form injections might only be required 2-3 times peryear. A steroid could also be added to the injection solution to reduceinflammation resulting from the injection itself.

[0987] Within another aspect of the present invention, methods areprovided for treating or preventing neovascular glaucoma, comprising thestep of administering to a patient a therapeutically effective amount ofa polynucleotide, polypeptide, antagonist and/or agonist to the eye,such that the formation of blood vessels is inhibited. In oneembodiment, the compound may be administered topically to the eye inorder to treat or prevent early forms of neovascular glaucoma. Withinother embodiments, the compound may be implanted by injection into theregion of the anterior chamber angle. Within other embodiments, thecompound may also be placed in any location such that the compound iscontinuously released into the aqueous humor. Within another aspect ofthe present invention, methods are provided for treating or preventingproliferative diabetic retinopathy, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eyes, such that theformation of blood vessels is inhibited.

[0988] Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

[0989] Within another aspect of the present invention, methods areprovided for treating or preventing retrolental fibroplasia, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eye, such that the formation of blood vessels is inhibited. Thecompound may be administered topically, via intravitreous injectionand/or via intraocular implants.

[0990] Additionally, diseases, disorders, and/or conditions which can betreated, prevented, and/or diagnosed with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

[0991] Moreover, diseases, disorders, and/or conditions and/or states,which can be treated, prevented, and/or diagnosed with thepolynucleotides, polypeptides, agonists and/or agonists include, but arenot limited to, solid tumors, blood born tumors such as leukemias, tumormetastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,rheumatoid arthritis, psoriasis, ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, and uvietis, delayed woundhealing, endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0992] In one aspect of the birth control method, an amount of thecompound sufficient to block embryo implantation is administered beforeor after intercourse and fertilization have occurred, thus providing aneffective method of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

[0993] Polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may be incorporated into surgical sutures in order toprevent stitch granulomas.

[0994] Polynucleotides, polypeptides, agonists and/or agonists may beutilized in a wide variety of surgical procedures. For example, withinone aspect of the present invention a compositions (in the form of, forexample, a spray or film) may be utilized to coat or spray an area priorto removal of a tumor, in order to isolate normal surrounding tissuesfrom malignant tissue, and/or to prevent the spread of disease tosurrounding tissues. Within other aspects of the present invention,compositions (e.g., in the form of a spray) may be delivered viaendoscopic procedures in order to coat tumors, or inhibit angiogenesisin a desired locale. Within yet other aspects of the present invention,surgical meshes which have been coated with anti-angiogenic compositionsof the present invention may be utilized in any procedure wherein asurgical mesh might be utilized. For example, within one embodiment ofthe invention a surgical mesh laden with an anti-angiogenic compositionmay be utilized during abdominal cancer resection surgery (e.g.,subsequent to colon resection) in order to provide support to thestructure, and to release an amount of the anti-angiogenic factor.

[0995] Within further aspects of the present invention, methods areprovided for treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

[0996] Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

[0997] The polynucleotides, polypeptides, agonists and/or agonists ofthe present invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

[0998] Lighter “d group” transition metals include, for example,vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species.Such transition metal species may form transition metal complexes.Suitable complexes of the above-mentioned transition metal speciesinclude oxo transition metal complexes.

[0999] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate and vanadyl complexes. Suitablevanadate complexes include metavanadate and orthovanadate complexes suchas, for example, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

[1000] Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten(IV) oxide andtungsten(VI) oxide. Suitable oxo molybdenum complexes include molybdate,molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexesinclude ammonium molybdate and its hydrates, sodium molybdate and itshydrates, and potassium molybdate and its hydrates Suitable molybdenumoxides include molybdenum(VI) oxide, molybdenum(VI) oxide, and molybdicacid. Suitable molybdenyl complexes include, for example, molybdenylacetylacetonate. Other suitable tungsten and molybdenum complexesinclude hydroxo derivatives derived from, for example, glycerol,tartaric acid, and sugars.

[1001] A wide variety of other anti-angiogenic factors may also beutilized within the context of the present invention. Representativeexamples include platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4): 1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

[1002] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated, prevented, and/ordiagnosed by the polynucleotides or polypeptides and/or antagonists oragonists of the invention, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

[1003] Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or polypeptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[1004] Diseases associated with increased apoptosis that could betreated, prevented, and/or diagnosed by the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, includeAIDS; neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Wound Healing and Epithelial Cell Proliferation

[1005] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of. dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe invention, may be clinically useful in stimulating wound healingincluding surgical wounds, excisional wounds, deep wounds involvingdamage of the dermis and epidermis, eye tissue wounds, dental tissuewounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitusulcers, arterial ulcers, venous stasis ulcers, burns resulting from heatexposure or chemicals, and other abnormal wound healing conditions suchas uremia, malnutrition, vitamin deficiencies and complicationsassociated with systemic treatment with steroids, radiation therapy andantineoplastic drugs and antimetabolites. Polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to promote dermal reestablishment subsequent to dermal loss.

[1006] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to increase the adherence ofskin grafts to a wound bed and to stimulate re-epithelialization fromthe wound bed. The following are a non-exhaustive list of grafts thatpolynucleotides or polypeptides, agonists or antagonists of theinvention, could be used to increase adherence to a wound bed:autografts, artificial skin, allografts, autodermic graft, autoepidermicgrafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplasticgrafts, cutis graft, delayed graft, dermic graft, epidermic graft,fascia graft, full thickness graft, heterologous graft, xenograft,homologous graft, hyperplastic graft, lamellar graft, mesh graft,mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,pedicle graft, penetrating graft, split skin graft, thick split graft.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, can be used to promote skin strength and to improve theappearance of aged skin.

[1007] It is believed that the polynucleotides or polypeptides, and/oragonists or antagonists of the invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

[1008] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could also be used to reduce the sideeffects of gut toxicity that result from radiation, chemotherapytreatments or viral infections. The polynucleotides or polypeptides,and/or agonists or antagonists of the invention, may have acytoprotective effect on the small intestine mucosa. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, mayalso stimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

[1009] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could further be used in full regenerationof skin in full and partial thickness skin defects, including bums,(i.e., repopulation of hair follicles, sweat glands, and sebaceousglands), treatment of other skin defects such as psoriasis. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could also be used to treat gastric anddoudenal ulcers and help heal by scar formation of the mucosal liningand regeneration of glandular mucosa and duodenal mucosal lining morerapidly. Inflamamatory bowel diseases, such as Crohn's disease andulcerative colitis, are diseases which result in destruction of themucosal surface of the small or large intestine, respectively. Thus, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to promote the resurfacing of the mucosalsurface to aid more rapid healing and to prevent progression ofinflammatory bowel disease. Treatment with the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, isexpected to have a significant effect on the production of mucusthroughout the gastrointestinal tract and could be used to protect theintestinal mucosa from injurious substances that are ingested orfollowing surgery. The polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used to treat diseasesassociate with the under expression of the polynucleotides of theinvention.

[1010] Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and bums, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

[1011] The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

[1012] In addition, the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to maintain the islet function so as to alleviate, delay orprevent permanent manifestation of the disease. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used as an auxiliary in islet cell transplantationto improve or promote islet cell function.

Neurological Diseases

[1013] Nervous system diseases, disorders, and/or conditions, which canbe treated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB 12 deficiency, folic acid deficiency, Wernicke disease,tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primarydegeneration of the corpus callosum), and alcoholic cerebellardegeneration; (7) neurological lesions associated with systemic diseasesincluding, but not limited to, diabetes (diabetic neuropathy, Bell'spalsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8)lesions caused by toxic substances including alcohol, lead, orparticular neurotoxins; and (9) demyelinated lesions in which a portionof the nervous system is destroyed or injured by a demyelinating diseaseincluding, but not limited to, multiple sclerosis, humanimmunodeficiency virus-associated myelopathy, transverse myelopathy orvarious etiologies, progressive multifocal leukoencephalopathy, andcentral pontine myelinolysis.

[1014] In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

[1015] The compositions of the invention which are useful for treatingor preventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

[1016] In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

[1017] A polypeptide or polynucleotide and/or agonist or antagonist ofthe present invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

[1018] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated, prevented, and/or diagnosed bya polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

[1019] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention include, but not limited to, include, but not limitedto, the following Gram-Negative and Gram-positive bacteria and bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis,Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucellosis,Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella(e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

[1020] Moreover, parasitic agents causing disease or symptoms that canbe treated, prevented, and/or diagnosed by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, the following families or class: Amebiasis,Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine,Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,Plasmodium virax, Plasmodium falciparium, Plasmodium malariae andPlasmodium ovale). These parasites can cause a variety of diseases orsymptoms, including, but not limited to: Scabies, Trombiculiasis, eyeinfections, intestinal disease (e.g., dysentery, giardiasis), liverdisease, lung disease, opportunistic infections (e.g., AIDS related),malaria, pregnancy complications, and toxoplasmosis polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedtotreat, prevent, and/or diagnose any of these symptoms or diseases. Inspecific embodiments, polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnosemalaria.

[1021] Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

[1022] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[1023] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[1024] Moreover, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase regeneration of tissuesdifficult to heal. For example, increased tendon/ligament regenerationwould quicken recovery time after damage. A polynucleotide orpolypeptide and/or agonist or antagonist of the present invention couldalso be used prophylactically in an effort to avoid damage. Specificdiseases that could be treated, prevented, and/or diagnosed include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

[1025] Similarly, nerve and brain tissue could also be regenerated byusing a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention to proliferate and differentiate nerve cells.Diseases that could be treated, prevented, and/or diagnosed using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic diseases, disorders, and/orconditions (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated, prevented, and/or diagnosed using thepolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention.

Chemotaxis

[1026] A polynucleotide. or polypeptide and/or agonist or antagonist ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[1027] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treat, prevent,and/or diagnose inflammation, infection, hyperproliferative diseases,disorders, and/or conditions, or any immune system disorder byincreasing the number of cells targeted to a particular location in thebody. For example, chemotaxic molecules can be used to treat, prevent,and/or diagnose wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treat,prevent, and/or diagnose wounds.

[1028] It is also contemplated that a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may inhibitchemotactic activity. These molecules could also be used to treat,prevent, and/or diagnose diseases, disorders, and/or conditions. Thus, apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention could be used as an inhibitor of chemotaxis.

Binding Activity

[1029] A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors),orsmall molecules.

[1030] Preferably, the molecule is closely related to the natural ligandof the polypeptide, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic. (See, Coliganet al., Current Protocols in Immunology 1(2):Chapter 5 (1991).)Similarly, the molecule can be closely related to the natural receptorto which the polypeptide binds, or at least, a fragment of the receptorcapable of being bound by the polypeptide (e.g., active site). In eithercase, the molecule can be rationally designed using known techniques.

[1031] Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

[1032] The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

[1033] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

[1034] Preferably, an ELISA assay can measure polypeptide level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure polypeptide level oractivity by either binding, directly or indirectly, to the polypeptideor by competing with the polypeptide for a substrate.

[1035] Additionally, the receptor to which a polypeptide of theinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

[1036] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[1037] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[1038] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

[1039] Other preferred fragments are biologically active fragments ofthe polypeptides of the invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

[1040] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and 3[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[1041] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the receptor is measured and the ability ofthe compound to bind to the receptor and elicit a second messengerresponse is measured to determine if the compound is a potential agonistor antagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

[1042] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (c) determining if a biological activity of thepolypeptide has been altered.

[1043] Also, one could identify molecules bind a polypeptide of theinvention experimentally by using the beta-pleated sheet regionscontained in the polypeptide sequence of the protein. Accordingly,specific embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, theamino acid sequence of each beta pleated sheet regions in a disclosedpolypeptide sequence. Additional embodiments of the invention aredirected to polynucleotides encoding polypeptides which comprise, oralternatively consist of, any combination or all of contained in thepolypeptide sequences of the invention. Additional preferred embodimentsof the invention are directed to polypeptides which comprise, oralternatively consist of, the amino acid sequence of each of the betapleated sheet regions in one of the polypeptide sequences of theinvention. Additional embodiments of the invention are directed topolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions in one of thepolypeptide sequences of the invention.

Targeted Delivery

[1044] In another embodiment, the invention provides a method ofdelivering compositions to targeted cells expressing a receptor for apolypeptide of the invention, or cells expressing a cell bound form of apolypeptide of the invention.

[1045] As discussed herein, polypeptides or antibodies of the inventionmay be associated with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions. In one embodiment, the invention provides amethod for the specific delivery of compositions of the invention tocells by administering polypeptides of the invention (includingantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[1046] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

[1047] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

[1048] Further contemplated is the use of the polypeptides of thepresent invention, or the polynucleotides encoding these polypeptides,to screen for molecules which modify the activities of the polypeptidesof the present invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

[1049] This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

[1050] Thus, the present invention provides methods of screening fordrugs or any other agents which affect activities mediated by thepolypeptides of the present invention. These methods comprise contactingsuch an agent with a polypeptide of the present invention or a fragmentthereof and assaying for the presence of a complex between the agent andthe polypeptide or a fragment thereof, by methods well known in the art.In such a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

[1051] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to thepolypeptides of the present invention, and is described in great detailin European Patent Application 84/03564, published on Sep. 13, 1984,which is incorporated herein by reference herein. Briefly stated, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with polypeptides of the present inventionand washed. Bound polypeptides are then detected by methods well knownin the art. Purified polypeptides are coated directly onto plates foruse in. the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

[1052] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptides of the present invention specifically compete with a testcompound for binding to the polypeptides or fragments thereof. In thismanner, the antibodies are used to detect the presence of any peptidewhich shares one or more antigenic epitopes with a polypeptide of theinvention.

[1053] The human LTRPC3 polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a LTRPC3 polypeptide,or a bindable peptide fragment, of this invention, comprising providinga plurality of compounds, combining the LTRPC3 polypeptide, or abindable peptide fragment, with each of a plurality of compounds for atime sufficient to allow binding under suitable conditions and detectingbinding of the LTRPC3 polypeptide or peptide to each of the plurality oftest compounds, thereby identifying the compounds that specifically bindto the LTRPC3 polypeptide or peptide.

[1054] Methods of identifying compounds that modulate the activity ofthe novel human LTRPC3 polypeptides and/or peptides are provided by thepresent invention and comprise combining a potential or candidatecompound or drug modulator of immunoglobulin biological activity with anLTRPC3 polypeptide or peptide, for example, the LTRPC3 amino acidsequence as set forth in SEQ ID NOS:2, 41, or 43, and measuring aneffect of the candidate compound or drug modulator on the biologicalactivity of the LTRPC3 polypeptide or peptide. Such measurable effectsinclude, for example, physical binding interaction; the ability tocleave a suitable immunoglobulin substrate; effects on native and clonedLTRPC3-expressing cell line; and effects of modulators or otherimmunoglobulin-mediated physiological measures.

[1055] Another method of identifying compounds that modulate thebiological activity of the novel LTRPC3 polypeptides of the presentinvention comprises combining a potential or candidate compound or drugmodulator of a immunoglobulin biological activity with a host cell thatexpresses the LTRPC3 polypeptide and measuring an effect of thecandidate compound or drug modulator on the biological activity of theLTRPC3 polypeptide. The host cell can also be capable of being inducedto express the LTRPC3 polypeptide, e.g., via inducible expression.Physiological effects of a given modulator candidate on the LTRPC3polypeptide can also be measured. Thus, cellular assays for particularimmunoglobulin modulators may be either direct measurement orquantification of the physical biological activity of the LTRPC3polypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a LTRPC3polypeptide as described herein, or an overexpressed recombinant LTRPC3polypeptide in suitable host cells containing an expression vector asdescribed herein, wherein the LTRPC3 polypeptide is expressed,overexpressed, or undergoes upregulated expression.

[1056] Another aspect of the present invention embraces a method ofscreening for a compound that is capable of modulating the biologicalactivity of a LTRPC3 polypeptide, comprising providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a LTRPC3 polypeptide, or a functional peptide or portionthereof (e.g., SEQ ID NOS:2, 41, or 43); determining the biologicalactivity of the expressed LTRPC3 polypeptide in the absence of amodulator compound; contacting the cell with the modulator compound anddetermining the biological activity of the expressed LTRPC3 polypeptidein the presence of the modulator compound. In such a method, adifference between the activity of the LTRPC3 polypeptide in thepresence of the modulator compound and in the absence of the modulatorcompound indicates a modulating effect of the compound.

[1057] Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as immunoglobulin modulators can be any small chemicalcompound, or biological entity (e.g., protein, sugar, nucleic acid,lipid). Test compounds will typically be small chemical molecules andpeptides. Generally, the compounds used as potential modulators can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

[1058] High throughput screening methodologies are particularlyenvisioned for the detection of modulators of the novel LTRPC3polynucleotides and polypeptides described herein. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds so identified can serve as conventional lead compounds, orcan themselves be used as potential or actual therapeutics.

[1059] A combinatorial chemical library is a collection of diversechemical compounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[1060] The preparation and screening of combinatorial chemical librariesis well known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

[1061] Devices for the preparation of combinatorial libraries arecommercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A AppliedBiosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.).In addition, a large number of combinatorial libraries are commerciallyavailable (e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Russia;Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., and thelike).

[1062] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell or tissueexpressing an ion channel is attached to a solid phase substrate. Insuch high throughput assays, it is possible to screen up to severalthousand different modulators or ligands in a single day. In particular,each well of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

[1063] In another of its aspects, the present invention encompassesscreening and small molecule (e.g., drug) detection assays which involvethe detection or identification of small molecules that can bind to agiven protein, i.e., a LTRPC3 polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

[1064] In such binding-based detection, identification, or screeningassays, a functional assay is not typically required. All that is neededis a target protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

[1065] An example of such an assay is the fluorescence based thermalshift assay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

[1066] To purify a LTRPC3 polypeptide or peptide to measure a biologicalbinding or ligand binding activity, the source may be a whole celllysate that can be prepared by successive freeze-thaw cycles (e.g., oneto three) in the presence of standard protease inhibitors. The LTRPC3polypeptide may be partially or completely purified by standard proteinpurification methods, e.g., affinity chromatography using specificantibody described infra, or by ligands specific for an epitope tagengineered into the recombinant LTRPC3 polypeptide molecule, also asdescribed herein. Binding activity can then be measured as described.

[1067] Compounds which are identified according to the methods providedherein, and which modulate or regulate the biological activity orphysiology of the LTRPC3 polypeptides according to the present inventionare a preferred embodiment of this invention. It is contemplated thatsuch modulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novel LTRPC3polypeptides by administering to an individual in need of such treatmenta therapeutically effective amount of the compound identified by themethods described herein.

[1068] In addition, the present invention provides methods for treatingan individual in need of such treatment for a disease, disorder, orcondition that is mediated by the LTRPC3 polypeptides of the invention,comprising administering to the individual a therapeutically effectiveamount of the LTRPC3-modulating compound identified by a method providedherein.

Antisense and Ribozyme (Antagonists)

[1069] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, 3, 5, 7, 9 and/or 11, or the complementary strand thereof,and/or to nucleotide sequences contained a deposited clone. In oneembodiment, antisense sequence is generated internally by the organism,in another embodiment, the antisense sequence is separately administered(see, for example, O° C.onnor, Neurochem., 56:560 (1991).Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Triple helix formation is discussed in, for instance, Lee et al.,Nucleic Acids Research, 6:3073 (1979); Cooney et al., Science, 241:456(1988); and Dervan et al., Science, 251:1300 (1991). The methods arebased on binding of a polynucleotide to a complementary DNA or RNA.

[1070] For example, the use of c-myc and c-myb antisense RNA constructsto inhibit the growth of the non-lymphocytic leukemia cell line HL-60and other cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoRI site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10mM MgCl2, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoRI/HindIII site of the retroviral vector PMV7 (WO 91/15580).

[1071] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[1072] In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

[1073] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[1074] Antisense oligonucleotides may be single or double stranded.Double stranded RNA's may be designed based upon the teachings ofPaddison et al., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); andInternational Publication Nos. WO 01/29058, and WO 99/32619; which arehereby incorporated herein by reference.

[1075] SiRNA reagents are specifically contemplated by the presentinvention. Such reagents are useful for inhibiting expression of thepolynucleotides of the present invention and may have therapeuticefficacy. Several methods are known in the art for the therapeutictreatment of disorders by the administration of siRNA reagents. One suchmethod is described by Tiscornia et al (PNAS, 100(4):1844-1848 (2003));WO0409769, filed July 18^(th), 2003; and Reich S J et al., Mol Vis. 2003May 30;9:210-6, which are incorporated by reference herein in itsentirety.

[1076] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

[1077] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[1078] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5.-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[1079] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[1080] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[1081] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-O-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[1082] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

[1083] While antisense nucleotides complementary to the coding regionsequence of the invention could be used, those complementary to thetranscribed untranslated region are most preferred.

[1084] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science,247:1222-1225 (1990). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[1085] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[1086] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[1087] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[1088] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[1089] The antagonist/agonist may also be employed to treat, prevent,and/or diagnose the diseases described herein.

[1090] Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present irivention,and/or (b) a ribozyme directed to the polynucleotide of the presentinvention.

Biotic Associations

[1091] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith other organisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

[1092] The mechanism by which a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase the hostorganisms ability, either directly or indirectly, to initiate and/ormaintain biotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

[1093] In an alternative embodiment, a polynucleotide or polypeptideand/or agonist or antagonist of the present invention may decrease thehost organisms ability to form biotic associations with anotherorganism, either directly or indirectly. The mechanism by which apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may decrease the host organisms ability, eitherdirectly or indirectly, to initiate and/or maintain biotic associationswith another organism is variable, though may include, modulatingosmolarity to undesirable levels, modulating pH to undesirable levels,modulating secretions of organic acids, modulating the secretion ofspecific proteins, phenolic compounds, nutrients, or the decreasedexpression of a protein required for host-biotic organisms interactions(e.g., a receptor, ligand, etc.). Additional mechanisms are known in theart and are encompassed by the invention (see, for example, “MicrobialSignalling and Communication”, eds., R. England, G. Hobbs, N. Bainton,and D. McL. Roberts, Cambridge University Press, Cambridge, (1999);which is hereby incorporated herein by reference).

[1094] The hosts ability to maintain biotic associations with aparticular pathogen has significant implications for the overall healthand fitness of the host. For example, human hosts have symbiosis withenteric bacteria in their gastrointestinal tracts, particularly in thesmall and large intestine. In fact, bacteria counts in feces of thedistal colon often approach 10¹² per milliliter of feces. Examples ofbowel flora in the gastrointestinal tract are members of theEnterobacteriaceae, Bacteriodes, in addition to a-hemolyticstreptococci, E. coli, Bifobacteria, Anaerobic cocci, Eubacteria,Costridia, lactobacilli, and yeasts. Such bacteria, among other things,assist the host in the assimilation of nutrients by breaking down foodstuffs not typically broken down by the hosts digestive system,particularly in the hosts bowel. Therefore, increasing the hosts abilityto maintain such a biotic association would help assure proper nutritionfor the host.

[1095] Aberrations in the enteric bacterial population of mammals,particularly humans, has been associated with the following disorders:diarrhea, ileus, chronic inflammatory disease, bowel obstruction,duodenal diverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

[1096] The composition of the intestinal flora, for example, is basedupon a variety of factors, which include, but are not limited to, theage, race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

[1097] Although the predominate intestinal flora comprises anaerobicorganisms, an underlying percentage represents aerobes (e.g., E. coli).This is significant as such aerobes rapidly become the predominateorganisms in intraabdominal infections—effectively becomingopportunistic early in infection pathogenesis. As a result, there is anintrinsic need to control aerobe populations, particularly for immunecompromised individuals.

[1098] In a preferred embodiment, a polynucleotides and polypeptides,including agonists, antagonists, and fragments thereof, are useful forinhibiting biotic associations with specific enteric symbiont organismsin an effort to control the population of such organisms.

[1099] Biotic associations occur not only in the gastrointestinal tract,but also on an in the integument. As opposed to the gastrointestinalflora, the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M.sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P.acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

[1100] Aberrations in the cutaneous flora are associated with a numberof significant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

[1101] Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

Pheromones

[1102] In another embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may increase theorganisms ability to synthesize, release, and/or respond to a pheromone,either directly or indirectly. Such a pheromone may, for example, alterthe organisms behavior and/or metabolism.

[1103] A polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones, either directly or indirectly. Preferably, any of thepheromones, and/or volatiles released from the organism, or induced, bya polynucleotide or polypeptide and/or agonist or antagonist of theinvention have behavioral effects on the organism.

[1104] For example, recent studies have shown that administration ofpicogram quantities of androstadienone, the most prominent androstenepresent on male human axillary hair and on the male axillary skin, tothe female vomeronasal organ resulted in a significant reduction ofnervousness, tension and other negative feelings in the femalerecipients (Grosser-B I, et al., Psychoneuroendocrinology, 25(3): 289-99(2000)).

Other Activities

[1105] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[1106] The polypeptide may also be employed for treating wounds due toinjuries, bums, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[1107] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat, prevent, and/or diagnoseneuronal damage which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS-related complex. The polypeptide of the invention mayhave the ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

[1108] The polypeptide of the invention may also be employed to maintainorgans before transplantation or for supporting cell culture of primarytissues.

[1109] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[1110] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also increase or decrease thedifferentiation or proliferation of embryonic stem cells, besides, asdiscussed above, hematopoietic lineage.

[1111] The polypeptide or polynucleotides and/or agonist or antagonistsof the present invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

[1112] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

[1113] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[1114] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

[1115] Also preferred is a method of treatment of an individual in needof an increased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

[1116] Polypeptide or polynucleotides and/or agonist or antagonists ofthe present invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

[1117] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

REFERENCES

[1118] Baldini G. , Mccarthy E. E. , Koticha D. K. Plasma membranetargeting of SNAP-25 increases its local concentration and is necessaryfor SNARE complex formation and regulated exocytosis. J. Cell Sci. 115:3341-3351 (2002).

[1119] Caterina, M. J., Leffler, A, Malmberg, A. B., Martin, W. J.,Trafton, J, Petersen-Zeitz, K. R., Koltzenburg, M, Basbaum, A. I. &Julius, D. Impaired nociception and pain sensation in mice lacking thecapsaicin receptor. Science. 288, 306-313 (2000).

[1120] Caterina, M. J., Rosen, T. A., Tominaga, M., Brake, A. J. &Julius, D. A capsaicin-receptor homologue with a high threshold fornoxious heat. Nature. 398, 436-41 (1999).

[1121] Duncan, L. M., Deeds, J., Hunter, J., Shao, J., Holmgren, L. M.,Woolf, E. A., Tepper, R. I. & Shyjan, A. W. Down-regulation of the novelgene melastatin correlates with potential for melanoma metastasis.Cancer Res. 58, 1515-1520 (1998).

[1122] Eddy S. Profile hidden Markov models. Bioinformatics 14:755-763(1998).

[1123] Freichel, M., Suh, S. H., Pfeifer, A., Schweig, U., Trost, C.,Weissgerber, P., Biel, M., Philipp, S., Freise, D., Droogmans, G.,Hofmann, F., Flockerzi, V. & Nilius, B. Lack of an endothelialstore-operated Ca2+ current impairs agonist-dependent vasorelaxation inTRP4−/− mice. Nat. Cell Biol. 3, 121-127 (2001).

[1124] Harteneck, C., Plant T. D. & Schultz, G. From worm to man: threesubfamilies of TRP channels. Trends Neurosci. 23, 159-166 (2000).

[1125] Inoue, R., Okada, T., Onoue, H., Hara, Y., Shimizu, S., Naitoh,S., Ito, Y. & Mori, Y. The transient receptor potential proteinhomologue TRP6 is the essential component of vascularalpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel. CircRes. 88, 325-332 (2001).

[1126] Lee N, Chen J, Wu S, Sun L, Huang M, Levesque P C, Rich A, FederJ N, Gray, K R, Lin J H, Janovitz E B, Blanar M A. Expression andcharacterization of human TRPM3. J Biol Chem. (2003).

[1127] Liman, E. R., Corey, D. P. & Dulac, C. TRP2: a candidatetransduction channel for mammalian pheromone sensory signaling. ProcNatl Acad Sci USA. 96, 5791-5796 (1999).

[1128] Missiaen, L., Robberecht, W., van den Bosch, L., Callewaert, G.,Parys, J. B., Wuytack, F., Raeymaekers, L., Nilius, B., Eggermont, J. &De Smedt, H. Abnormal intracellular Ca²⁺ homeostasis and disease. CellCalcium. 28, 1-21 (2000).

[1129] Nagamine, K., Kudoh, J., Minoshima, S., Kawasaki, K., Asakawa,S., Ito F. & Shimizu, N. Molecular cloning of a novel putative Ca²⁺channel protein (TRPC7) highly expressed in brain. Genomics 54, 124-131(1998).

[1130] Peng, J. B., Chen, X. Z., Berger, U. V., Vassilev, P. M.,Tsukaguchi, H., Brown, E. M. & Hediger, M. A. Molecular cloning andcharacterization of a channel-like transporter mediating intestinalcalcium absorption. J. Biol. Chem. 274, 22739-22746 (1999).

[1131] Prawitt, D., Enklaar, T., Klemm, G., Gartner, B., Spangenberg,C., Winterpacht, A., Higgins, M., Pelletier, J. & Zabel, B.Identification and characterization of MTR1, a novel gene with homologyto melastatin (MLSN1) and the trp family located in the BWS-WT2 criticalregion on chromosome 11p15.5 and showing allele-specific expression. HumMol Genet. 9, 203-16 (2000).

[1132] Radu A, Moore M S, Blobel G. The peptide repeat domain ofnucleoporin Nup98 functions as a docking site in transport across thenuclear pore complex. Cell 81:215-222 (1995).

[1133] Runnels, L. W., Yue, L. & Clapham, D. E. TRP-PLIK, a bifunctionalprotein kinase and ion channel activities. Science 291, 1043-1047(2001).

[1134] Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G. &Plant, T. D. OTRPC4, a nonselective cation channel that conferssensitivity to extracellular osmolarity. Nat. Cell Biol. 2, 695-702(2000).

[1135] Tsavaler, L., Shapero, M. H., Morkowski, S. & Laus R. TRP-P8, anovel prostate-specific gene, is up-regulated in prostate cancer andother malignancies and shares high homology with transient receptorpotential calcium channel proteins. Cancer Res. 61, 3760-3769 (2001).

[1136] Walker, R. G., Willingham, A. T. & Zuker, C. S. A Drosophilamechanosensory transduction channel. Science. 287, 2229-34 (2000).

[1137] Xu, S. Z. & Beech, D. J. TrpC1 is a membrane-spanning subunit ofstore-operated Ca²⁺ channels in native vascular smooth muscle. Circ Res.88, 84-7 (2001).

[1138] Yue, L., Peng, J. B., Hediger, M. A., Clapham, D. E. CaT1manifests the pore properties of the calcium-release-activated calciumchannel. Nature. 410, 705-709 (2001).

[1139] Zygmunt, P. M., Petersson, J., Andersson, D. A., Chuang, H.,Sorgard, M., Di Marzo V., Julius, D. & Hogestatt, E. D. Vanilloidreceptors on sensory nerves mediate the vasodilator action ofanandamide. Nature. 400, 452-457 (1999).

EXAMPLES Description of the Preferred Embodiments Example 1 Method Usedto Identify the Novel LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k,LTRPC3l Polynucleotides of the Present Invention—Bioinformatics Analysis

[1140] The polynucleotide sequence of the LTRPC3 variant (SEQ ID NO:14;co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002; InternationalPublication No. WO 03/012063, published Feb. 13, 2003) was used as aprobe to BLAST the GenBank human cDNA database using TBLASTN programmRNA sequence gi|AL136545 (SEQ ID NO:106) was found to further extendthe 5′ end of the LTRPC3 variant polynucleotide sequence (SEQ ID NO:14)by an addition of 534 bases. The nucleotide sequence of this 5′extension is provided as SEQ ID NO:107. The new elongated 5′ end of theLTRPC3 variant was mapped to the human genomic sequence (NCBI version31) using software BLIS (Biotique Local Integration System). These 534bases represent 3 exons in the 5′ upstream region of the original TRPM3(i.e., LTRPC3) gene (see FIG. 20).

[1141] Protein domain analysis of the new 5′ end of the LTRPC3 variantusing HMMSEARCH (Eddy S.) against the Pfam protein domain databasesuggests that this portion of the extended LTRPC3 variant proteincontains one NSF domain and one Nucleoporin_FG domain, as shown in FIG.21. NSF domain was proposed to function in regulation of intracellulartraffic via fusion of two lipid bilayers (Baldini et al.). Our previouspublication(Lee et al.) on the LTRPC3 variant suggest the LTRPC3 variantis a membrane protein with storage-operated Ca²⁺ channel activity. TheNSF domain on the LTRPC3 variant protein may function to guide thelocalization of the LTRPC3 variant carrying intracellular membrane.Nucleoporin_FG repeats are found in diverse nucleoporins (Radu et al.).It has been suggested that these repeats mediate interactions withsubstrates to be transported through the nuclear pore. The function ofNucleoporin_FG domain in the LTRPC3 variant is unknown.

[1142] The extended polynucleotide sequence of the gi|AL136545 sequence(SEQ ID NO:106) was used to extend the coding region of the LTRPC3polynucleotide sequence (SEQ ID NO:14) which resulted in the LTRPC3gvariant of the present invention (SEQ IDNO:1). The same sequence wasalso used to extend the LTRCP3b, LTRCP3c, LTRCP3d, LTRCP3e, and LTRCP3fsequences as well which resulted in the LTRPC3h (SEQ IDNO:3), LTRPC3l(SEQ IDNO:5), LTRPC3j (SEQ IDNO:7), LTRPC3k (SEQ IDNO:9), and LTRPC3l(SEQ IDNO:) variants of the present invention.

[1143] The full-length polynucleotide sequences of the LTRPC3g (SEQIDNO:1), LTRPC3h (SEQ IDNO:3), LTRPC3l (SEQ IDNO:5), LTRPC3j (SEQIDNO:7), LTRPC3k (SEQ IDNO:9), and LTRPC3l (SEQ IDNO:11) variants areprovided in FIGS. 1A-E, FIGS. 2A-F, FIGS. 3A-F, FIGS. 4A-F, FIGS. 5A-F,and FIGS. 6A-F, respectively.

Example 2 Cloning the Novel Transient Receptor Potential Channel Member,LTRPC3

[1144] A number of methods may be employed to clone the full-lengthpolynucleotides of the present invention. Specifically, thepolynucleotides of the present invention may be PCR amplified using theprimers provided below. The oligonucleotide primers may be used used toamplify fragments from the human kidney Marathon-Ready cDNA library(Clontech). The reaction mixture in 50 ul containing 5 ul cDNA library,0.5 mM each primer, 5 mM dNTPs (1.25 mM each), 5 ul of 10×PCR and 0.5unit of TaqPlus Precision polymerase (Stratagene) can be used. Thereaction can be repeated for 30 cycles (94° C. for 45 seconds, 55° C.for 45 seconds, and 72° C. for 4 minutes). The amplified fragments canthen be cloned into the sequencing vector pCR4 Blunt-TOPO (Invitrogen)for sequence analysis. Primer Name Primer Sequence TRPM3-1CATGGGGAAGAAGTGGAGGGATGCGG (SEQ ID NO: 169) TRPM3-2CGCTCAGAAATCCTGGATAGAAAGAG (SEQ ID NO: 170) 86-N2-NCTGTATCAAAAGATACTCGCACATACAT (SEQ ID NO: 171)

[1145] The TRPM3-1C and TRPM3-2C are forward primers. Either of thelatter could be used in conjunction with the reverse 86-N2-NC primer toclone the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3lpolynucleotides of the present invention.

Example 3 Expression Profiling of the Transient Receptor PotentialChannel Member. LTRPC3

[1146] The following protocol was used to elucidate the expressionprofile of the LTRPC3 polypeptide (SEQ ID NO:15) and is described inco-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to elucidate the expression profile of theLTRPC3g-l variants of the present invention using primer pairs specificfor these variants. The LTRPC3 expression profile is provided here forrepresentative purposes only although it is expected that the LTRPC3g-lvariants will have similar expression patterns as the LTRPC3 polypeptidesince the LTRPC3g-l variants represent longer N-terminally extendedversions of LTRPC3.

RT-PCR

[1147] A PCR primer pair was designed to measure the steady state levelsof the LTRPC3 mRNA by quantitative RT-PCR. LTRP6.tp1sCGCAGCTGGAAGACCTTATC (SEQ ID NO: 109) LTRP6.tp1a AAGCTGCTCTGACGGACAAT(SEQ ID NO: 110)

[1148] Briefly, first strand cDNA was made from commercially availablemRNA. The relative amount of cDNA used in each assay was determined byperforming a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. The cyclophilinprimer pair detected small variations in the amount of cDNA in eachsample and these data were used for normalization of the data obtainedwith the primer pair for the LTRPC3 transcript. The PCR data wasconverted into a relative assessment of the difference in transcriptabundance amongst the tissues tested and the data is presented in FIG.8. Transcripts corresponding to the LTRPC3 transcript were found to behighly expressed in the kidney; and significantly in spinal cord,testis, and brain.

Northern Blot Analysis

[1149] Human tissue Northern blots (Clontech) were probed with an RNAprobe derived from a 645-bp DNA fragment amplified from the primer pair86-1-C (SEQ ID NO:111) and 86-5-NC (5′-AGGGAAGGGGAAGTGGTTGATCTC-3′, SEQID NO:112). Hybridization of the blot was performed at 68° C. inExpressHyb (Clontech) for 6 hours, with 1×10⁶ cpm/ml of ³²P-labeledprobe. Autoradiography was performed for 1 week at −70° C.

[1150] The results of the Northern hybridization are shown in FIG. 9. Asshown, Transcripts corresponding to the LTRPC3 transcript were found tobe highly expressed in kidney, and to a lesser extent in brain, andtestis.

Example 4 Method of Assessing the Expression Profile of the LTRPC3Polypeptides of the Present Invention Using Expanded mRNA Tissue andCell Sources

[1151] The following protocol was used to elucidate the expandedexpression profile of the LTRPC3 polypeptide (SEQ ID NO:15) and isdescribed in co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to elucidate the expanded expressionprofile of the LTRPC3g-l variants of the present invention using primerpairs specific for these variants. The expanded LTRPC3 expressionprofile is provided here for representative purposes only although it isexpected that the LTRPC3g-l variants have similar expanded expressionpatterns as the LTRPC3 polypeptide since the LTRPC3g-l variantsrepresent N-terminally extended versions of LTRPC3.

[1152] Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

[1153] The specific sequence to be measured was aligned with relatedgenes found in GenBank to identity regions of significant sequencedivergence to maximize primer and probe specificity. Gene-specificprimers and probes were designed using the ABI primer express softwareto amplify small amplicons (150 base pairs or less) to maximize thelikelihood that the primers function at 100% efficiency. Allprimer/probe sequences were searched against Public Genbank databases toensure target specificity. Primers and probes were obtained from ABI.

[1154] For LTRPC3, the primer probe sequences were as follows ForwardPrimer 5′-TCAGAGAATGGGCCAACAAGA-3′ (SEQ ID NO: 113) Reverse Primer5′-CGAAAACGCTCGAGGAATGA-3′ (SEQ ID NO: 114) TaqMan Probe5′-CAGGCCTAGGTTCCTCCTCTCGGAAA-3′ (SEQ ID NO: 115)

DNA Contamination

[1155] To access the level of contaminating genomic DNA in the RNA, theRNA was divided into 2 aliquots and one half was treated with Rnase-freeDnase (Invitrogen). Samples from both the Dnase-treated and non-treatedwere then subjected to reverse transcription reactions with (RT+) andwithout (RT−) the presence of reverse transcriptase. TaqMan assays werecarried out with gene-specific primers (see above) and the contributionof genomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

[1156] 100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500 μM of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

[1157] Quantitative sequence detection was carried out on an ABI PRISM7700 by adding to the reverse transcribed reaction 2.5 μM forward andreverse primers, 500 μM of each dNTP, buffer and 5 U AmpliTaq Gold™. ThePCR reaction was then held at 94° C. for 12 min, followed by 40 cyclesof 94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

[1158] The threshold cycle (Ct) of the lowest expressing tissue (thehighest Ct value) was used as the baseline of expression and all othertissues were expressed as the relative abundance to that tissue bycalculating the difference in Ct value between the baseline and theother tissues and using it as the exponent in 2^((Δct))

[1159] The expanded expression profile of the LTRPC3 polypeptide isprovided in FIG. 12 and 13 and described elsewhere herein.

Example 5 Complementary Oligonucleotides to the LTRPC3 Polynucleotide

[1160] The following protocol was used to elucidate the association ofthe LTRPC3 polypeptide (SEQ ID NO:15) to regulation of FEN1 and isdescribed in co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to demonstrate an association to theregulation of FEN1 for the LTRPC3g-l variants of the present inventionusing antisense oligos specific for these variants. The FEN1associationdata for LTRPC3 is provided here for representative purposes onlyalthough it is expected that the LTRPC3g-l variants have a similar FEN1association as the LTRPC3 polypeptide since the LTRPC3g-l variantsrepresent N-terminally extended versions of LTRPC3.

[1161] Antisense molecules or nucleic acid sequences complementary tothe LTRPC3 protein-encoding sequence, or any part thereof, was used todecrease or to inhibit the expression of naturally occurring LTRPC3.Although the use of antisense or complementary oligonucleotidescomprising about 15 to 35 base-pairs is described, essentially the sameprocedure is used with smaller or larger nucleic acid sequencefragments. An oligonucleotide based on the coding sequence of LTRPC3protein as depicted in SEQ ID NO:14, for example, is used to inhibitexpression of naturally occurring LTRPC3. The complementaryoligonucleotide is typically designed from the most unique 5′ sequenceand is used either to inhibit transcription by preventing promoterbinding to the coding sequence, or to inhibit translation by preventingthe ribosome from binding to the LTRPC3 protein-encoding transcript.However, other regions may also be targeted.

[1162] Using an appropriate portion of a 5′ sequence of SEQ ID NO:14, aneffective antisense oligonucleotide includes any of about 15-35nucleotides spanning the region which translates into the signal or 5′coding sequence, among other regions, of the polypeptide as shown in SEQID NO:15. Appropriate oligonucleotides are designed using OLIGO 4.06software and the LTRPC3 protein coding sequence (SEQ ID NO:14).Preferred oligonucleotides are deoxynucleotide, or chimericdeoxynucleotide/ribonucleotide based and are provided below. Theoligonucleotides were synthesized using chemistry essentially asdescribed in U.S. Pat. No. 5,849,902; which is hereby incorporatedherein by reference in its entirety. !ID#? Sequence 15737CCAUGGACAGAGAUGAGAAGCUUGGU (SEQ ID NO: 119) 15738AGUGGUCCCGUUGUCAGCCAGAAUGU (SEQ ID NO: 120) 15739CCUUCCACUAUGAGUGCCACCACAGU (SEQ ID NO: 121) 15740GUGUCCUUCUGAUCCCAUCCGAAAUU (SEQ ID NO: 122) 15741UGGUAUGGCCGGACAACAUCUCUUCU (SEQ ID NO: 123)

[1163] The LTRPC3 polypeptide has been shown to be involved in theregulation of mammalian base-excision repair. Subjecting cells with aneffective amount of a pool of all five of the above antisenseoligoncleotides resulted in a significant increase in FEN1expression/activity providing convincing evidence that LTRPC3 at leastregulates the activity and/or expression of FEN1 either directly, orindirectly. Moreover, the results suggest that LTRPC3 is involved in thenegative regulation of FEN1 activity and/or expression, either directlyor indirectly. The FEN1 assay used is described below and was based uponthe analysis of FEN1 activity as a downstream marker for proliferativesignal transduction events.

Transfection of Post-quiescent A549 Cells with AntiSenseOligonucleotides Materials Needed

[1164] A549 cells maintained in DMEM with high glucose (Gibco-BRL)supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and 1×penicillin/streptomycin.

[1165] Opti-MEM (Gibco-BRL)

[1166] Lipofectamine 2000 (Invitrogen)

[1167] Antisense oligomers (Sequitur)

[1168] Polystyrene tubes.

[1169] Tissue culture treated plates.

[1170] Quiescent cells were prepared as follows:

[1171] Day 0: 300, 000 A549 cells were seeded in a T75 tissue cultureflask in 10 ml of A549 media (as specified above), and incubated in at37° C., 5% CO₂ in a humidified incubator for 48 hours.

[1172] Day 2: The T75 flasks were rocked to remove any loosely adherentcells, and the A549 growth media removed and replenished with 10 ml offresh A549 media. The cells were cultured for six days without changingthe media to create a quiescent cell population.

[1173] Day 8: Quiescent cells were plated in multi-well format andtransfected with antisense oligonucleotides.

[1174] A549 cells were transfected according to the following:

[1175] 1. Trypsinize T75 flask containing quiescent population of A549cells.

[1176] 2. Count the cells and seed 24-well plates with 60K quiescentA549 cells per well.

[1177] 3. Allow the cells to adhere to the tissue culture plate(approximately 4 hours).

[1178] 4. Transfect the cells with antisense and controloligonucleotides according to the following:

[1179] a. A 10× stock of lipofectamine 2000 (10 ug/ml is 10×) wasprepared, and diluted lipid was allowed to stand at RT for 15 minutes.

[1180] b. Stock solution of lipofectamine 2000 was 1 mg/ml.

[1181] c. 10× solution for transfection was 10 ug/ml.

[1182] To prepare 10× solution, dilute 10 ul of lipofectamine 2000 stockper 1 ml of Opti-MEM (serum free media).

[1183] a. A 10× stock of each oligomer was prepared to be used in thetransfection.

[1184] b. Stock solutions of oligomers were at 100 uM in 20 mM HEPES, pH7.5.

[1185] c. 10× concentration of oligomer was 0.25 uM.

[1186] To prepare the 10× solutions, dilute 2.5 ul of oligomer per 1 mlof Opti-MEM.

[1187] a. Equal volumes of the 10× lipofectamine 2000 stock and the 10×oligomer solutions were mixed well, and incubated for 15 minutes at RTto allow complexation of the oligomer and lipid. The resulting mixturewas 5×.

[1188] b. After the 15 minute complexation, 4 volumes of full growthmedia was added to the oligomer/lipid complexes (solution was 1×).

[1189] c. The media was aspirated from the cells, and 0.5 ml of the 1×oligomer/lipid complexes added to each well.

[1190] d. The cells were incubated for 16-24 hours at 37° C. in ahumidified CO₂ incubator.

[1191] e. Cell pellets were harvested for RNA isolation and TaqMananalysis of downstream marker genes.

TaqMan Reactions

[1192] Quantitative RT-PCR analysis was performed on total RNA prepsthat had been treated with DNaseI or poly A selected RNA. The Dnasetreatment may be performed using methods known in the art, thoughpreferably using a Qiagen Rneasy kit to purify the RNA samples, whereinDNAse I treatment is performed on the column.

[1193] Briefly, a master mix of reagents was prepared according to thefollowing table: Dnase I Treatment Reagent Per r'xn (in uL) 10x Buffer2.5 Dnase I (1 unit/ul @ 1 unit per ug sample) 2 DEPC H₂O 0.5 RNA sample@ 0.1 ug/ul 20 (2-3 ug total) Total 25

[1194] Next, 5 ul of master mix was aliquoted per well of a 96-well PCRreaction plate (PE part # N801-0560). RNA samples were adjusted to 0.1ug/ul with DEPC treated H₂O (if necessary), and 20 ul was added to thealiquoted master mix for a final reaction volume of 25 ul.

[1195] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and briefly spun in a plate centrifuge(Beckman) to collect all volume in the bottom of the tubes. Generally, ashort spin up to 500 rpm in a Sorvall RT is sufficient.

[1196] The plates were incubated at 37° C. for 30 mins. Then, an equalvolume of 0.1 mM EDTA in 10 mM Tris was added to each well, and heatinactivated at 70° C. for 5 min. The plates were stored at −80° C. uponcompletion.

RT Reaction

[1197] A master mix of reagents was prepared according to the followingtable: RT reaction RT No RT Reagent Per Rx'n (in ul) Per Rx'n (in ul)10x RT buffer 5 2.5 MgCl₂ 11 5.5 DNTP mixture 10 5 Random Hexamers 2.51.25 Rnase inhibitors 1.25 0.625 RT enzyme 1.25 — Total RNA 500 ng (100ng no RT) 19.0 max 10.125 max DEPC H₂O — — Total 50 uL 25 uL

[1198] Samples were adjusted to a concentration so that 500 ng of RNAwas added to each RT rx′n (10 ng for the no RT). A maximum of 19 ul canbe added to the RT rx′n mixture (10.125 ul for the no RT.) Any remainingvolume up to the maximum values was filled with DEPC treated H₂O, sothat the total reaction volume was 50 ul (RT) or 25 ul (no RT).

[1199] On a 96-well PCR reaction plate (PE part # N801-0560), 37.5 ul ofmaster mix was aliquoted (22.5 ul of no RT master mix), and the RNAsample added for a total reaction volume of 50 ul (25 ul, no RT).Control samples were loaded into two or even three different wells inorder to have enough template for generation of a standard curve.

[1200] The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and spin briefly in a plate centrifuge(Beckman) to collect all volume in the bottom of the tubes. Generally, ashort spin up to 500 rpm in a Sorvall RT is sufficient.

[1201] For the RT-PCR reaction, the following thermal profile was used:

[1202] 25° C. for 10 min

[1203] 48° C. for 30 min

[1204] 95° C. for 5 min

[1205] 4° C. hold (for 1 hour)

[1206] Store plate @ −20° C. or lower upon completion.

[1207] TaqMan reaction (Template comes from RT plate.)

[1208] A master mix was prepared according to the following table:TaqMan reaction (per well) Reagent Per Rx'n (in ul) TaqMan Master Mix4.17 100 uM Probe (SEQ ID NO: 118) .025 100 uM Forward primer (SEQ IDNO: 116) .05 100 uM Reverse primer (SEQ ID NO: 117) .05 Template — DEPCH₂O 18.21 Total 22.5

[1209] The primers used for the RT-PCR reaction is as follows: FEN1primer and probes Forward Primer: CCACCTGATGGGCATGTTCT (SEQ ID NO: 116)Reverse Primer: CGGCTTGCCATCAAAGACATA (SEQ ID NO: 117) TaqMan Probe:CCGCACCATTCGCATGATGGAG (SEQ ID NO: 118)

[1210] Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix wasaliquouted per well of a 96-well optical plate. Then, using P-10pipetter, 2.5 ul of sample was added to individual wells. Generally, RTsamples are run in triplicate with each primer/probe set used, and no RTsamples are run once and only with one primer/probe set, often gapdh (orother internal control).

[1211] A standard curve is then constructed and loaded onto the plate.The curve has five points plus one no template control (NTC, =DEPCtreated H₂O). The curve was made with a high point of 50 ng of sample(twice the amount of RNA in unknowns), and successive samples of 25, 10,5, and 1 ng. The curve was made from a control sample(s) (see above).

[1212] The wells were capped using optical strip well caps (PE part #N801-0935), placed in a plate, and spun in a centrifuge to collect allvolume in the bottom of the tubes. Generally, a short spin up to 500 rpmin a Sorvall RT is sufficient.

[1213] Plates were loaded onto a PE 5700 sequence detector making surethe plate is aligned properly with the notch in the upper right handcorner. The lid was tightened down and run using the 5700 and 5700quantitation programes and the SYBR probe using the following thermalprofile:

[1214] 50° C. for 2 min

[1215] 95° C. for 10 min

[1216] and the following for 40 cycles:

[1217] 95° C. for 15 sec

[1218] 60° C. for 1 min

[1219] Change the reaction volume to 25 ul.

[1220] Once the reaction was complete, a manual threshold of around 0.1was set to minimuze the background signal.Additional informationrelative to operation of the GeneAmp 5700 machine may be found inreference to the following manuals: “GeneAmp 5700 Sequence DetectionSystem Operator Training CD”; and the “User's Manual for 5700 SequenceDetection System”; available from Perkin-Elmer and hereby incorporatedby reference herein in their entirety.

Example 6 Method of Assessing the Cellular Localization of the LTRPC3Polypeptide

[1221] The following protocol was used to confirm the cellularlocalization of the LTRPC3 polypeptide (SEQ ID NO:15) and is describedin co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to demonstrate the cellular localizationof the LTRPC3g-l variants of the present invention. The cellularlocalization data for LTRPC3 is provided here for representativepurposes only although it is expected that the LTRPC3g-l variants arealso localized to the cellular membrane since the LTRPC3g-l variantsrepresent N-terminally extended versions of LTRPC3.

[1222] HEK 293 cells were cultured in Dulbecco's modified mediumcontaining 10% heat-inactivated fetal bovine serum and grown on thepoly-D-lysine-coated glass coverslips. The cells were transientlytransfected with the pcDNA3.1/Hygro-LTRPC3-HA vector with Fugene(Roche). Forty-eight hours later, cells were fixed with 4%paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, blockedin PBS containing 5% FBS and 5% normal goat serum, and stained with 10μg/ml Fluorescein-conjugated anti-HA High Affinity antibody (3F10,Roche) and DAPI (0.5 μg/ml; Molecular Probes). Immunostained cellcultures were examined using a laser scanning confocal microscope (ZEISSLSM510). A 63× oil immersion objective was used with appropriate filtersets. Images were reconstructed from confocal stacks of Z-series scansof 10-20 optical sections through a depth of 5-15 μM.

[1223] The cellular localization of HA-tagged LTRPC3 was exclusivelydetected at the subplasma membrane region of the transfected cells (seeFIG. 18), which is consistent with LTRPC3 being an integral membraneprotein. Under these conditions approximately ˜70% of cells wereexpressing LTRPC3.

Example 7 Method of Assessing the Ion Channel Activity of the LTRPC3Polypeptide

[1224] The following protocol was used to demonstrate the putative ionchannel activity of the LTRPC3 polypeptide (SEQ ID NO:15) and isdescribed in co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to demonstrate the putative ion channelactivity of the LTRPC3g-l variants of the present invention. The ionchannel activity data for LTRPC3 is provided here for representativepurposes only since it is expected that the LTRPC3g-l variants also havethe same or similar ion channel activity as LTRPC3 since the LTRPC3g-lvariants represent N-terminally extended versions of LTRPC3.

[1225] The cytoplasmic Ca²⁺ indicator Fluo-4-AM (Molecular Probe) andthe Fluorometric Imaging Plate Reader (FLIPR™, Molecular Devices)instrument were used to detect changes in intracellular Ca²⁺. ThepcDNA3.1/Hygro-LTRPC3-HA transfected cells were seeded on PDL-coated96-well plates at a density of 70,000 cells/well 24 hours aftertransfection and used 24 hours after plating. Transfected cells wereloaded with 4 lM Fluo-4-AM at 37° C. for 30 min in a nominally Ca²⁺-freeor 1 mM CaCl₂ buffer containing 140 mM NaCl, 4.7 mM KCl, 1 mM MgCl₂, 10mM HEPES, 10 mM Glucose, and 2.5 mM Probenecid (Sigma), pH 7.4.Extracellular Fluo-4-AM was removed and cells were maintained in eitherCa²⁺-free buffer or buffer containing 1 mM Ca²⁺ at room temperatureprior to the experiments, which were conducted within 30 min after dyeremoval. Fluo-4 was excited at 488 nm using an argon laser and emittedlight was selected using a 510-570 nm bandpass filter. Baselineintracellular fluorescence was established during the initial 50 secondsof the FLIPR read, then 1, 3 or 10 mM Ca²⁺ was added to each well andsubsequent changes in the intracellular Ca²⁺ were monitored for 8minutes. For store-depletion studies, 2 μM thapsigargin or 100 μMcarbachol was added to Fluo-4-loaded cells in Ca²⁺-free buffer beforeadding Ca²⁺ on FLIPR. For pharmacology studies, 100 μM LaCl₃ or 100 μMGdCl₃ were added to Fluo-4-loaded cells in 1 mM Ca²⁺ buffer beforeadding Ca²⁺ on FLIPR. Experiments were carried out at room temperature.The results of these physiometric studies are shown in FIGS. 18 and 19.

[1226] The addition of Ca²⁺ to the media resulted in aconcentration-dependent influx of Ca²⁺ into LTRPC3-expressing cells (asshown in FIG. 18, right panels), indicating that LTRPC3 is a functionalCa²⁺ channel. In contrast, vector-transfected cells showed minimal Ca²⁺influx under the same experimental conditions (as shown in FIG. 18, leftpanels). The non-transfected cells were indistinguishable from thevector-transfected cells (data not shown). Therefore, LTRPC3 is aconstitutively active channel capable of mediating Ca²⁺ influx.

[1227] To further address the mechanism of LTRPC3-mediated Ca²⁺ entry,similar Ca²⁺ addition experiments were performed on transfected cellsincubated (˜30 min) in a nominally Ca²⁺-free solution. Previous studieshave shown that lowering extracellular Ca²⁺ concentration belowphysiological levels can deplete intracellular Ca²⁺ stores in many celltypes including HEK 293 (EMBO J. 17, 4274-4282 (1998)). Incubatingvector-transfected HEK 293 cells in a nominally Ca²⁺-free solution gaverise to Ca²⁺ entry that was dependent on the concentration of Ca²⁺ addedto the buffers, indicating Ca²⁺ influx was mediated through endogenousSOCs in HEK293 cells (as shown in FIG. 19; left panel). In LTRPC3 cells,the Ca²⁺ transients triggered by similar Ca²⁺ treatment were much larger(as shown in FIG. 19, right panel). This Ca²⁺ entry observed in LTRPC3cells incubated in Ca²⁺-free media were greater than in 1 mM Ca²⁺ media,indicating that LTRPC3-mediated Ca²⁺ entry can be potentiated by thestore-depletion.

Example 8 Method of Assessing the Expression Profile of the LTRPC3Polypeptide Using In Situ Hybridization

[1228] The following protocol was used to demonstrate the expressionprofile of the LTRPC3 polypeptide (SEQ ID NO:15) using In situhybridization and is described in co-pending U.S. Ser. No. 10/210,152,filed Aug. 1, 2002 and International Publication No. WO 03/012063,published Feb. 13, 2003. The same protocol may be employed todemonstrate the In situ hybridization expression profile of theLTRPC3g-l variants of the present invention using appropriate primerpairs. The In situ hybridization expression data for LTRPC3 is providedhere for representative purposes only since it is expected that theLTRPC3g-l variants also have the same or similar In situ hybridizationexpression profile as LTRPC3 since the LTRPC3g-l variants representN-terminally extended versions of LTRPC3.

[1229] Human Kidney was collected and received from the National DiseaseResearch Interchange (Philadelphia, Pa.) according to IRB approvedprotocol. Tissue sections were embedded in OCT compound (Miles) andsnap-frozen by immersion in 2-methylbutane cooled in dry ice andsubsequently stored at −70° C.

[1230] Templates for LTRPC3 cRNA probes were derived from a 678-bpLTRPC3 fragment, cloned in a pCR-BluntII-TOPO vector (Invitrogen)utilizing the primer pair: (forward: 5′-CAGCTGGAAGACCTTATCGGG-3′ (SEQ IDNO:124); reverse: 5′-TGGGAGGTGGGTGTAGTCTGAAGA-3′ (SEQ ID NO:125)). Thetemplate for positive control cRNA human lysozyme probe was derived froma 638 bp EST (Incyte Genomics, GenBank Accession No:AA588081).³⁵S-labeled riboprobes were synthesized via in vitro transcriptionutilizing the Riboprobe® Combination System (Promega) where T7 and Sp6RNA polymerase yielded sense and antisense probes respectively forLTRPC3 while T7 and T3 RNA polymerases yielded antisense and senseprobes respectively for human lysozyme. Cryostat tissue sections cut at10 μm and fixed in 4.0% formalin were used for in situ hybridization aspreviously described (Dambach, D. M., et al., (2002) Hepatology 35,1093-1103.): Briefly, tissue sections were acetylated; dehydrated in agraded ethanol series; immersed in chloroform; alcohol rinsed; air driedand then hybridized with sense and antisense ³⁵S RNA probes (1.5×10⁶cpm/slide) for 16-20 hours at 60° C. Following hybridization, slideswere rinsed in 4×SSC/50% formamide and 4×SSC; treated with RNAse A (20μg/ml; Invitrogen) at 37° C.; washed through increasing stringentsolutions to final high stringency wash in 0.1×SSC at 60° C.;dehydrated; air dried and then coated with NTB-2 emulsion (EastmanKodak). Slides were placed in a dark box with desiccant at 4° C. anddeveloped after one and four weeks exposure. Sections were stained withhematoxylin and eosin, and coverslipped. Expression signals weredetected by dark phase microscopy. Cellular phenotype identification wasby bright field microscopy.

[1231] The In situ hybridization results of the LTRPC3 polypeptide isprovided in FIG. 17 and described elsewhere herein.

Example 9 Method of Further Assessing Cellular Localization of theLTRPC3 Polypeptide

[1232] The following protocol was used to further assess the cellularlocalization of the LTRPC3 polypeptide (SEQ ID NO:15) and is describedin co-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to further assess the cellularlocalization of the LTRPC3g-l variants of the present invention. Thecellular localization data for LTRPC3 is provided here forrepresentative purposes only since it is expected that the LTRPC3g-lvariants are also localized to the cellular membrane since the LTRPC3g-lvariants represent N-terminally extended versions of LTRPC3.

[1233] HEK 293 cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 10% heat-inactivated fetal bovine serum and grown onpoly-D-lysine-coated (PDL) glass coverslips. The cells were transientlytransfected with hLTRPC3-HA with FuGENE 6 (Roche MolecularBiochemicals). Forty-eight hours later, cells were stained in culturemedia with the membrane probe VybrantTm CM-DiI (5 μl/ml; MolecularProbes) at 37° C. for 5 min and 4° C. for 15 min. After washing withPBS, cells were fixed with 4% paraformaldehyde in PBS, permeabilizedwith 0.1% Triton X-100, blocked in PBS containing 5% FBS and 5% normalgoat serum, and stained with 10 μg/ml Fluorescein-conjugated anti-HAHigh Affinity antibody (3F10; Roche Molecular Biochemicals) and DAPI(0.5 μg/ml; Molecular Probes). Immunostained cell cultures were examinedusing a laser scanning confocal microscope (ZEISS LSM510), a 63× oilimmersion objective, and appropriate filter sets. Images shown are of asingle optical section approximately 1 μm thick.

[1234] The expanded confocal microscopy results of the LTRPC3polypeptide is provided in FIG. 18 and described elsewhere herein.

Example 10 Method of Further Assessing the Ion Channel Activity of theLTRPC3 Polypeptide

[1235] The following protocol was used to further assess the ion channelactivity of the LTRPC3 polypeptide (SEQ ID NO:15) and is described inco-pending U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 andInternational Publication No. WO 03/012063, published Feb. 13, 2003. Thesame protocol may be employed to further assess the ion channel activityof the LTRPC3g-l variants of the present invention. The ion channelactivity data for LTRPC3 is provided here for representative purposesonly since it is expected that the LTRPC3g-l variants also have the sameor similar ion channel activity as LTRPC3 since the LTRPC3g-l variantsrepresent N-terminally extended versions of LTRPC3.

[1236] The cytoplasmic Ca²⁺ indicator Fluo-4-AM (Molecular Probes) and aFluorometric Imaging Plate Reader (FLIPR™; Molecular Devices) instrumentwere used to detect changes in intracellular Ca²⁺ concentration. ThehLTRPC3-transfected cells were seeded on PDL-coated 96-well plates at adensity of 70,000 cells/well 24 hours after transfection and used 24hours after plating. Cells were loaded with 4 μM Fluo-4-AM at 37° C. for30 min in a nominally Ca²⁺-free or 1 mM CaCl₂ buffer containing (in mM):140 NaCl, 4.7 KCl, 1 MgCl₂, 10 HEPES, 10 Glucose, and 2.5 Probenecid(Sigma), pH 7.4. Extracellular Fluo-4-AM was removed and cells weremaintained in either Ca²⁺-free buffer or buffer containing 1 mM Ca²⁺ atroom temperature prior to the experiments, which were conducted within30 min after dye removal. Fluo-4 was excited at 488 nm using an argonlaser and emitted light was selected using a 510-570 nm bandpass filter.Baseline intracellular fluorescence was established during the initial50 seconds of the FLIPR read, then 1, 3, or 10 mM Ca²⁺ was added to eachwell and subsequent changes in the intracellular Ca²⁺ were monitored for8 minutes. For store-depletion or receptor activation studies, 2 μMthapsigargin or 50 μM carbachol, respectively, was added toFluo-4-loaded cells in Ca²⁺-free buffer before adding 2 mM Ca²⁺ onFLIPR. For pharmacology studies, 100 μM GdCl₃ was added to Fluo-4-loadedcells in 0 or 1 mM Ca²⁺ buffer, as described in herein, prior to thestart of the FLIPR recordings. Experiments were carried out at roomtemperature.

[1237] The results of the expanded physiometric experiments are providedin FIG. 19 and described herein.

Example 11 Method of Assessing the Putative Kinase Activity of theLTRPC3 Polypeptide

[1238] A number of methods may be employed to assess the potental kinaseactivity of the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, and LTRPC3lpolypeptides. One preferred method is described below. A fusionconstruct is made whereby the LTRPC3g-, LTRPC3h-, LTRPC3I-, LTRPC3j-,LTRPC3k-, or LTRPC3l-encoding polynucleotide is operably linked to thecoding region of the HA protein. CHO-K1 or HEK-293 cells grown on 100-mmdishes are transiently transfected with 8 μg of novel LTRPC3(g, h, I, j,k, or l)-HA cDNA construct in the pTracer-CMV2 (Invitrogen) vector withLipofectAMINE 2000 (Gibco). Cells are harvested after 48 hours with 3 mlof RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% IGEPALCA-630, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, and 10 mMiodoacetamide]. LTRPC3(g, h, I, j, k, or l)-HA is immunoprecipitatedwith mouse monoclonal immunoglobulin G2a (IgG2a) HA probe (F-7) coupledto agarose (Santa Cruz Biotechnology). The agarose is sedimented andwashed three times with RIPA buffer, and 2×SDS sample buffer is added.The samples may be resolved by SDS-PAGE and Western blotting followingstandard methods. HA probe Y-11 antibody could be the primary antibody(Santa Cruz Biotechnology), and horseradish peroxidase-linked antibodyto rabbit Ig (Amersham Pharmacia Biotech) could be the secondaryantibody. The SuperSignal West Dura substrate may be used forchemiluminescent detection (Pierce)

[1239] For phosphorylation experiments, purified GST-kinase fusionproteins and mutants are incubated at 37° C. for 30 min in the presenceor absence of MBP as a test substrate in a 50-μl reaction. Thesereactions are performed in KIN buffer {50 mM Mops (pH 7.2), 100 mM NaCl,20 mM MgCl2, 0.5 mM ATP, and 2 μCi of [-32P]ATP}. Immunokinase reactionscontaining immunopurified LTRPC3(g, h, I, j, k, or l)-HA are incubatedat 37° C. for 30 min in a 50-μl reaction containing KIN buffer with 75mM n-octyl-D-glucopyranoside. The reactions are terminated by theaddition of 2×SDS sample buffer, and the proteins were resolved bySDS-PAGE and Coomassie staining for the GST-kinase experiment or bySDS-PAGE and Western blotting for the immunokinase assay. The gels aredried, and 32P incorporation is visualized by autoradiography for theGST-kinase experiment. For the immunokinase experiment, 32Pincorporation may be visualized by autoradiography of the transferredproteins on polyvinylidene difluoride membrane (Bio-Rad) before Westernblotting.

Example 12 Method of Assessing Ability of LTRPC3 Polypeptides toAssociate with Other Proteins Using the Yeast Two-hybrid System

[1240] In an effort to determine whether the LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l polypeptides of the present invention arecapable of functioning as an ion channel or kinase protein, it would beimportant to effectively test the interaction between LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l and various portions of otherproteins, particularly known ion channel proteins, for example, in ayeast two-hybrid system. Such a system could be created using methodsknown in the art (see, for example, S. Fields and O. Song, Nature,340:245-246 (1989); and Gaston-S M and Loughlin-K R, Urology, 53(4):835-42 (1999); which are hereby incorporated herein by reference intheir entirety, including the articles referenced therein).

[1241] Cytoplasmic NH and COOH terminal domains of different proteins,preferably ion channel proteins (such as those referenced herein), couldbe subcloned and expressed as fusion proteins of the GAL4 DNA binding(DB) domain using molecular biology techniques within the skill of theartisan.

[1242] Exemplary subunits which could be used in the two-hybrid systemto assess the ability of LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, orLTRPC3l to associate with other ion channel proteins include, but arenot limited to, the NH and/or C-terminal domain TRP1, TRP2, TRP3, TRP4,TRP5, TRP6, TRP7, signalling proteins, etc.

Example 13 Method of Assessing Ability of LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l Polypeptides to Form Oligomeric Complexeswith Itself or Other Ion Channel Proteins in Solution

[1243] Aside from determining whether the LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l polypeptides are capable of interactingwith other proteins, preferably ion channel proteins, in a yeasttwo-hybrid assay, it would be an important next step to assess itsability to form oligomeric complexes with itself, in addition to otherproteins, preferably ion channel proteins, in solution. Such a findingwould be significant as it would provide convincing evidence thatLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l could serve asan ion channel protein.

[1244] A number of methods could be used to that are known in the art,for example, the method described by Sanguinetti, M. C., et al., Nature,384:80-83 (1996) could be adapted using methods within the skill of theartisan.

Example 14 Method of Identifying the Cognate Ligand of the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l Polypeptide

[1245] A number of methods are known in the art for identifying thecognate binding partner of a particular polypeptide. For example, theencoding LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3lpolynucleotide could be engineered to comprise an epitope tag. Theepitope could be any epitope known in the art or disclosed elsewhereherein. Once created, the epitope tagged LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l encoding polynucleotide could be clonedinto an expression vector and used to transfect a variety of cell linesrepresenting different tissue origins (e.g., brain, testis, kindey,testis, liver, etc.). The transfected cell lines could then be inducedto overexpress the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, orLTRPC3l polypeptide. The presence of the LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l polypeptide on the cell surface could bedetermined by fractionating whole cell lysates into cellular andmembrane protein fractions and performing immunoprecipitation using theantibody directed against the epitope engineered into the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l polypeptide. Monoclonalor polyclonal antibodies directed against the LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l polypeptide could be created and used inplace of the antibodies directed against the epitope.

[1246] Alternatively, the cell surface proteins could be distinguishedfrom cellular proteins by biotinylating the surface proteins and thenperforming immunoprecipitations with antibody specific to the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l protein. Afterelectrophoretic separation, the biotinylated protein could be detectedwith streptavidin-HRP (using standard methods known to those skilled inthe art). Identification of the proteins bound to LTRPC3g, LTRPC3h,LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l could be made in those cells byimmunoprecipation, followed by one-dimensional electrophoresis, followedby various versions of mass spectrometry. Such mass-spectrometry methodsare known in the art, such as for example the methods taught byCiphergen Biosystems Inc. (see U.S. Pat. No. 5,792,664; which is herebyincorporated herein by reference).

Example 15 Isolation of a Specific Clone from the Deposited Sample

[1247] The deposited material in the sample assigned the ATCC DepositNumber cited in Table 1 for any given cDNA clone also may contain one ormore additional plasmids, each comprising a cDNA clone different fromthat given clone. Thus, deposits sharing the same ATCC Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table 1.Typically, each ATCC deposit sample cited in Table 1 comprises a mixtureof approximately equal amounts (by weight) of about 1-10 plasmid DNAs,each containing a different cDNA clone and/or partial cDNA clone; butsuch a deposit sample may include plasmids for more or less than 2 cDNAclones.

[1248] Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table 1.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:1, 3, 5, 8, 317 or 318.

[1249] Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2 nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

[1250] Alternatively, two primers of 17-20 nucleotides derived from bothends of the SEQ ID NO:1, 3, 5, 7, 9, or 11 (i.e., within the region ofSEQ ID NO:1, 3, 5, 7, 9, or 11 bounded by the 5′ NT and the 3′ NT of theclone defined in Table 1) are synthesized and used to amplify thedesired cDNA using the deposited cDNA plasmid as a template. Thepolymerase chain reaction is carried out under routine conditions, forinstance, in 25 ul of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v)gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94 degree C. for 1 min; annealing at 55 degree C. for 1 min;elongation at 72 degree C. for 1 min) are performed with a Perkin-ElmerCetus automated thermal cycler. The amplified product is analyzed byagarose gel electrophoresis and the DNA band with expected molecularweight is excised and purified. The PCR product is verified to be theselected sequence by subcloning and sequencing the DNA product.

[1251] The polynucleotide(s) of the present invention, thepolynucleotide encoding the polypeptide of the present invention, or thepolypeptide encoded by the deposited clone may represent partial, orincomplete versions of the complete coding region (i.e., full-lengthgene). Several methods are known in the art for the identification ofthe 5′ or 3′ non-coding and/or coding portions of a gene which may notbe present in the deposited clone. The methods that follow are exemplaryand should not be construed as limiting the scope of the invention.These methods include but are not limited to, filter probing, cloneenrichment using specific probes, and protocols similar or identical to5′ and 3′ “RACE” protocols that are well known in the art. For instance,a method similar to 5′ RACE is available for generating the missing 5′end of a desired full-length transcript. (Fromont-Racine et al., NucleicAcids Res. 21(7):1683-1684 (1993)).

[1252] Briefly, a specific RNA oligonucleotide is ligated to the 5′ endsof a population of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

[1253] This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA that may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

[1254] This modified RNA preparation is used as a template for firststrand cDNA synthesis using a gene specific oligonucleotide. The firststrand synthesis reaction is used as a template for PCR amplification ofthe desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

[1255] An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc.Nat'l.Acad.Sci.USA, 85:8998-9002 (1988). Briefly, acDNA clone missing either the 5′ or 3′ end can be reconstructed toinclude the absent base pairs extending to the translational start orstop codon, respectively. In some cases, cDNAs are missing the start oftranslation, therefor. The following briefly describes a modification ofthis original 5′ RACE procedure. Poly A+ or total RNAs reversetranscribed with Superscript II (Gibco/BRL) and an antisense or Icomplementary primer specific to the cDNA sequence. The primer isremoved from the reaction with a Microcon Concentrator (Amicon). Thefirst-strand cDNA is then tailed with dATP and terminal deoxynucleotidetransferase (Gibco/BRL). Thus, an anchor sequence is produced which isneeded for PCR amplification. The second strand is synthesized from thedA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), anoligo-dT primer containing three adjacent restriction sites (XhoIJ Sailand ClaI) at the 5′ end and a primer containing just these restrictionsites. This double-stranded cDNA is PCR amplified for 40 cycles with thesame primers as well as a nested cDNA-specific antisense primer. The PCRproducts are size-separated on an ethidium bromide-agarose gel and theregion of gel containing cDNA products the. predicted size of missingprotein-coding DNA is removed. cDNA is purified from the agarose withthe Magic PCR Prep kit (Promega), restriction digested with XhoI orSalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) atXhoI and EcoRV sites. This DNA is transformed into bacteria and theplasmid clones sequenced to identify the correct protein-coding inserts.Correct 5′ ends are confirmed by comparing this sequence with theputatively identified homologue and overlap with the partial cDNA clone.Similar methods known in the art and/or commercial kits are used toamplify and recover 3′ ends.

[1256] Several quality-controlled kits are commercially available forpurchase. Similar reagents and methods to those above are supplied inkit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of fulllength genes. A second kit is available from Clontech which is amodification of a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32(1991). The major differences in procedure are that the RNA isalkaline hydrolyzed after reverse transcription and RNA ligase is usedto join a restriction site-containing anchor primer to the first-strandcDNA. This obviates the necessity for the dA-tailing reaction whichresults in a polyT stretch that is difficult to sequence past.

[1257] An alternative to generating 5′ or 3′ cDNA from RNA is to usecDNA library double-stranded DNA. An asymmetric PCR-amplified antisensecDNA strand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to ObtainFull Length Genes

[1258] Once a gene of interest is identified, several methods areavailable for the identification of the 5′ or 3′ portions of the genewhich may not be present in the original cDNA plasmid. These methodsinclude, but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar and identical to 5′ and 3′ RACE.While the full-length gene may be present in the library and can beidentified by probing, a useful method for generating the 5′ or 3′ endis to use the existing sequence information from the original cDNA togenerate the missing information. A method similar to 5′ RACE isavailable for generating the missing 5′ end of a desired full-lengthgene. (This method was published by Fromont-Racine et al., Nucleic AcidsRes., 21(7): 1683-1684 (1993)). Briefly, a specific RNA oligonucleotideis ligated to the 5′ ends of a population of RNA presumably 30containing full-length gene RNA transcript and a primer set containing aprimer specific to the ligated RNA oligonucleotide and a primer specificto a known sequence of the gene of interest, is used to PCR amplify the5′ portion of the desired full length gene which may then be sequencedand used to generate the full length gene. This method starts with totalRNA isolated from the desired source, poly A RNA may be used but is nota prerequisite for this procedure. The RNA preparation may then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase if used is then inactivated and the RNA is treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNApreparation can then be used as a template for first strand cDNAsynthesis using a gene specific oligonucleotide. The first strandsynthesis reaction can then be used as a template for PCR amplificationof the desired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of theapoptosis related of interest. The resultant product is then sequencedand analyzed to confirm that the 5′ end sequence belongs to the relevantapoptosis related.

Example 16 Chromosomal Mapping of the Polynucleotides

[1259] An oligonucleotide primer set is designed according to thesequence at the 5′ end of SEQ ID NO:1, 3, 5, 7, 9, or 11. This primerpreferably spans about 100 nucleotides. This primer set is then used ina polymerase chain reaction under the following set of conditions: 30seconds, 95 degree C.; 1 minute, 56 degree C.; 1 minute, 70 degree C.This cycle is repeated 32 times followed by one 5 minute cycle at 70degree C. Mammalian DNA, preferably human DNA, is used as template inaddition to a somatic cell hybrid panel containing individualchromosomes or chromosome fragments (Bios, Inc). The reactions areanalyzed on either 8% polyacrylamide gels or 3.5% agarose gels.Chromosome mapping is determined by the presence of an approximately 100bp PCR fragment in the particular somatic cell hybrid.

Example 17 Bacterial Expression of a Polypeptide

[1260] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 15, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites, such as BamHI andXbaI, at the 5′ end of the primers in order to clone the amplifiedproduct into the expression vector. For example, BamHI and XbaIcorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vectorencodes antibiotic resistance (Ampr), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

[1261] The pQE-9 vector is digested with BamHI and XbaI and theamplified fragment is ligated into the pQE-9 vector maintaining thereading frame initiated at the bacterial RBS. The ligation mixture isthen used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) whichcontains multiple copies of the plasmid pREP4, that expresses the lacIrepressor and also confers kanamycin resistance (Kanr). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

[1262] Clones containing the desired constructs are grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.600) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalacto pyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

[1263] Cells are grown for an extra 3 to 4 hours. Cells are thenharvested by centrifugation (20 mins at 6000×g). The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidine HCl by stirringfor 3-4 hours at 4 degree C. The cell debris is removed bycentrifugation, and the supernatant containing the polypeptide is loadedonto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

[1264] Briefly, the supernatant is loaded onto the column in 6 Mguanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 Mguanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

[1265] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Example 18 Purification of a Polypeptide from an Inclution Body

[1266] The following alternative method can be used to purify apolypeptide expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 degree C.

[1267] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10 degree C. and the cellsharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[1268] The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[1269] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

[1270] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 degree C. without mixingfor 12 hours prior to further purification steps.

[1271] To clarify the refolded polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 um membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 nm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

[1272] Fractions containing the polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perceptive Biosystems) and weak anion (Poros CM-20, PerceptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A280 monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

[1273] The resultant polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Coomassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified protein can alsobe tested for endotoxin/LPS contamination, and typically the LPS contentis less than 0.1 ng/ml according to LAL assays.

Example 19 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

[1274] In this example, the plasmid shuttle vector pAc373 is used toinsert a polynucleotide into a baculovirus to express a polypeptide. Atypical baculovirus expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV) followed by convenient restriction sites, which may include,for example BamHI, XbaI and Asp718. The polyadenylation site of thesimian virus 40 (“SV40”) is often used for efficient polyadenylation.For easy selection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

[1275] Many other baculovirus vectors can be used in place of the vectorabove, such as pVL941 and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

[1276] A polynucleotide encoding a polypeptide of the present inventionis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ ends of the DNA sequence, as outlined in Example 15, tosynthesize insertion fragments. The primers used to amplify the cDNAinsert should preferably contain restriction sites at the 5′ end of theprimers in order to clone the amplified product into the expressionvector. Specifically, the cDNA sequence contained in the depositedclone, including the AUG initiation codon and the naturally associatedleader sequence identified elsewhere herein (if applicable), isamplified using the PCR protocol described in Example 15. If thenaturally occurring signal sequence is used to produce the protein, thevector used does not need a second signal peptide. Alternatively, thevector can be modified to include a baculovirus leader sequence, usingthe standard methods described in Summers et al., “A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555 (1987).

[1277] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

[1278] The plasmid is digested with the corresponding restrictionenzymes and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.).

[1279] The fragment and the dephosphorylated plasmid are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria containing the plasmid are identified by digesting DNAfrom individual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

[1280] Five ug of a plasmid containing the polynucleotide isco-transformed with 1.0 ug of a commercially available linearizedbaculovirus DNA (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.), using the lipofection method described by Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virusDNA and 5 ug of the plasmid are mixed in a sterile well of a microtiterplate containing 50 ul of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ulGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

[1281] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, supra. An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10.) After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

[1282] To verify the expression of the polypeptide, Sf9 cells are grownin Grace's medium supplemented with 10% heat-inactivated FBS. The cellsare infected with the recombinant baculovirus containing thepolynucleotide at a multiplicity of infection (“MOI”) of about 2. Ifradiolabeled proteins are desired, 6 hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). After 42 hours,5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham)are added. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[1283] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the produced protein.

Example 20 Expression of a Polypeptide in Mammalian Cells

[1284] The polypeptide of the present invention can be expressed in amammalian cell. A typical mammalian expression vector contains apromoter element, which mediates the initiation of transcription ofmRNA, a protein coding sequence, and signals required for thetermination of transcription and polyadenylation of the transcript.Additional elements include enhancers, Kozak sequences and interveningsequences flanked by donor and acceptor sites for RNA splicing. Highlyefficient transcription is achieved with the early and late promotersfrom SV40, the long terminal repeats (LTRs) from Retroviruses, e.g.,RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter).

[1285] Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0.Mammalian host cells that could be used include, human Hela, 293, H9 andJurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quailQC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

[1286] Alternatively, the polypeptide can be expressed in stable celllines containing the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

[1287] The transformed gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

[1288] A polynucleotide of the present invention is amplified accordingto the protocol outlined in herein. If the naturally occurring signalsequence is used to produce the protein, the vector does not need asecond signal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.) The amplifiedfragment is isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragmentthen is digested with appropriate restriction enzymes and again purifiedon a 1% agarose gel.

[1289] The amplified fragment is then digested with the same restrictionenzyme and purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then -ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

[1290] Chinese hamster ovary cells lacking an active DHFR gene is usedfor transformation. Five μg of an expression plasmid is cotransformedwith 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 21 Protein Fusions

[1291] The polypeptides of the present invention are preferably fused toother proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

[1292] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also should have convenient restrictionenzyme sites that will facilitate cloning into an expression vector,preferably a mammalian expression vector. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

[1293] The naturally occurring signal sequence may be used to producethe protein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.) Human IgG Fc region:GGGATCCGGAGCCCAAATCTTCTGACAAAACTCA (SEQ ID NO: 108)CACATGCCCACCGTGCCCAGCACCTGAATTCGAG GGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGA GGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 22 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, orLTRPC3l Polypeptide of the Present Invention

[1294] As described elsewhere herein, the present invention encompassesthe creation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to theLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l polypeptide ofthe present invention. A number of methods are available to one skilledin the art for creating such mutants. Such methods may include acombination of PCR amplification and gene cloning methodology. Althoughone of skill in the art of molecular biology, through the use of theteachings provided or referenced herein, and/or otherwise known in theart as standard methods, could readily create each deletion mutant ofthe present invention, exemplary methods are described below.

[1295] Briefly, using the isolated cDNA clone encoding the full-lengthLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l polypeptidesequence (as described in Example 15, for example), appropriate primersof about 15-25 nucleotides derived from the desired 5′ and 3′ positionsof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, orSEQ ID NO:11, may be designed to PCR amplify, and subsequently clone,the intended N- and/or C-terminal deletion mutant. Such primers couldcomprise, for example, an inititation and stop codon for the 5′ and 3′primer, respectively. Such primers may also comprise restriction sitesto facilitate cloning of the deletion mutant post amplification.Moreover, the primers may comprise additional sequences, such as, forexample, flag-tag sequences, kozac sequences, or other sequencesdiscussed and/or referenced herein.

[1296] For example, in the case of the L775 to T1709 LTRPC3g N-terminaldeletion mutant, the following primers could be used to amplify a cDNAfragment corresponding to this deletion mutant: 5′ Primer 5′-GCAGCAGCGGCCGC CTCAAG (SEQ ID NO: 126)             NotI GTAATTCTGGGAATTCTAC-3′ 3′ Primer 5′- GCAGCA GTCGAC GGTGTGC (SEQ ID NO: 127)             SalI TTGCTTTCAAAGCTTTGG -3′

[1297] For example, in the case of the M1 to N1150 LTRPC3g C-terminaldeletion mutant, the following primers could be used to amplify a cDNAfragment corresponding to this deletion mutant: 5′ Primer 5′- GCAGCAGCGGCCGC ATGTA (SEQ ID NO: 128)              NotI TGTGCGAGTATCTTTTG -3′3′ Primer 5′- GCAGCA GTCGAC GTTAAAG (SEQ ID NO: 129)             SalIACAGCAATGAGGAGGTTG -3′

[1298] The resulting C-terminal deletion mutant could be used as apotential, membrane bound, LTRPC3g decoy receptor.

[1299] For example, in the case of the M1 to N1216 LTRPC3g C-terminaldeletion mutant, the following primers could be used to amplify a cDNAfragment corresponding to this deletion mutant: 5′ Primer 5′- GCAGCAGCGGCCGC ATGTAT (SEQ ID NO: 130)              NotI GTGCGAGTATCTTTTG -3′3′ Primer 5′- GCAGCA GTCGAC ATTAGAT (SEQ ID NO: 131)             SalIGAGTTGAACCGATCATCC -3′

[1300] The resulting C-terminal deletion mutant could be used as apotential, membrane bound, LTRPC3g decoy receptor.

[1301] Representative PCR amplification conditions are provided below,although the skilled artisan would appreciate that other conditions maybe required for efficient amplification. A 100 ul PCR reaction mixturemay be prepared using 10 ng of the template DNA (cDNA clone of LTRPC3),200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA polymerase (PE), andstandard Taq DNA polymerase buffer. Typical PCR cycling condition are asfollows: 20-25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72degrees   1 cycle: 10 min, 72 degrees

[1302] After the final extension step of PCR, 5 U Klenow Fragment may beadded and incubated for 15 min at 30 degrees.

[1303] Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E.coli cells usingmethods provided herein and/or otherwise known in the art.

[1304] The 5′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula: (S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l gene (SEQ ID NO:1, 3, 5,7, 9, or 11), and ‘X’ is equal to the most N-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 5′ primer, while the second termwill provide the end 3′ nucleotide position of the 5′ primercorresponding to sense strand of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. Once the correspondingnucleotide positions of the primer are determined, the final nucleotidesequence may be created by the addition of applicable restriction sitesequences to the 5′ end of the sequence, for example. As referencedherein, the addition of other sequences to the 5′ primer may be desiredin certain circumstances (e.g., kozac sequences, etc.).

[1305] The 3′ primer sequence for amplifying any additional N-terminaldeletion mutants may be determined by reference to the followingformula: (S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to thenucleotide position of the initiating start codon of the LTRPC3g,LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l gene (SEQ ID NO:1, 3, 5,7, 9, or 11), and ‘X’ is equal to the most C-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 3′ primer, while the second termwill provide the end 3′ nucleotide position of the 3′ primercorresponding to the anti-sense strand of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 3′ primermay be desired in certain circumstances (e.g., stop codon sequences,etc.). The skilled artisan would appreciate that modifications of theabove nucleotide positions may be necessary for optimizing PCRamplification.

[1306] The same general formulas provided above may be used inidentifying the 5′ and 3′ primer sequences for amplifying any C-terminaldeletion mutant of the present invention. Moreover, the same generalformulas provided above may be used in identifying the 5′ and 3′ primersequences for amplifying any combination of N-terminal and C-terminaldeletion mutant of the present invention. The skilled artisan wouldappreciate that modifications of the above nucleotide positions may benecessary for optimizing PCR amplification.

Example 23 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

[1307] As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

[1308] Numerous methods have been developed to date for introducingforeign genes, either under the control of an inducible, constitutivelyactive, or endogenous promoter, into organisms. Of particular interestare the inducible promoters (see, M. Gossen, et al., Proc. Natl. Acad.Sci. USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc,.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

[1309] A new alternative method of controlling gene expression levels ofa protein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

[1310] Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

[1311] Fusion protein constructs are created using polynucleotidesequences of the present invention with one or more copies (preferablyat least 2, 3, 4, or more) of a conditional aggregation domain (CAD) adomain that interacts with itself in a ligand-reversible manner (i.e.,in the presence of an inducing agent) using molecular biology methodsknown in the art and discussed elsewhere herein. The CAD domain may bethe mutant domain isolated from the human FKBP12 (Phe³⁶ to Met) protein(as disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

[1312] The specific methods described herein for controlling proteinsecretion levels through controlled ER aggregation are not meant to belimiting are would be generally applicable to any of the polynucleotidesand polypeptides of the present invention, including variants,homologues, orthologs, and fragments therein.

Example 24 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

[1313] Many eukaryotic cell surface and proteins arepost-translationally processed to incorporate N-linked and O-linkedcarbohydrates (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem.54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838).Protein glycosylation is thought to serve a variety of functionsincluding: augmentation of protein folding, inhibition of proteinaggregation, regulation of intracellular trafficking to organelles,increasing resistance to proteolysis, modulation of proteinantigenicity, and mediation of intercellular adhesion (Fieldler andSimons (1995) Cell, 81:309-312; Helenius (1994) Mol. Biol. Of the Cell5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982)Cell, 37:417-427; Alexamnder and Elder (1984), Science, 226:1328-1330;and Flack et al., (1994), J. Biol. Chem. . . , 269:14015-14020). Inhigher organisms, the nature and extent of glycosylation can markedlyaffect the circulating half-life and bio-availability of proteins bymechanisms involving receptor mediated uptake and clearance (Ashwell andMorrell, (1974), Adv. Enzymol., 41:99-128; Ashwell and Harford (1982),Ann. Rev. Biochem., 51:531-54). Receptor systems have been identifiedthat are thought to play a major role in the clearance of serum proteinsthrough recognition of various carbohydrate structures on theglycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et al.,(1992), Arch. Biochem. Biophys., 298:49-55). Thus, production strategiesresulting in incomplete attachment of terminal sialic acid residuesmight provide a means of shortening the bioavailability and half-life ofglycoproteins. Conversely, expression strategies resulting in saturationof terminal sialic acid attachment sites might lengthen proteinbioavailability and half-life.

[1314] In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992),J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

[1315] Moreover, it is unclear to what extent a glycosylation site inone species will be recognized by another species glycosylationmachinery. Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E.coli, yeast, orviral organisms; or an E.coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

[1316] A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

Example 25 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention through Molecular Evolution

[1317] Although many of the most biologically active proteins known arehighly effective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

[1318] Thus, one aspect of the present invention relates to the abilityto enhance specific characteristics of invention through directedmolecular evolution. Such an enhancement may, in a non-limiting example,benefit the inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

[1319] For example, an engineered transient potential receptor may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered transient potential receptor may be constitutively activein the absence of ligand binding. In yet another example, an engineeredtransient potential receptor may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor transient potential receptor activation (e.g., ligand binding,phosphorylation, conformational changes, etc.). Such transient potentialreceptors would be useful in screens to identify transient potentialreceptor modulators, among other uses described herein.

[1320] Directed evolution is comprised of several steps. The first stepis to establish a library of variants for the gene or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

[1321] Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

[1322] Random mutagenesis has been the most widely recognized method todate. Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

[1323] While both of the aforementioned methods are effective forcreating randomized pools of macromolecule variants, a third method,termed “DNA Shuffling”, or “sexual PCR” (W P C, Stemmer, PNAS, 91:10747,(1994)) has recently been elucidated. DNA shuffling has also beenreferred to as “directed molecular evolution”, “exon-shuffling”,“directed enzyme evolution”, “in vitro evolution”, and “artificialevolution”. Such reference terms are known in the art and areencompassed by the invention. This new, preferred, method apparentlyovercomes the limitations of the previous methods in that it not onlypropagates positive traits, but simultaneously eliminates negativetraits in the resulting progeny.

[1324] DNA shuffling accomplishes this task by combining the principalof in vitro recombination, along with the method of “error-prone” PCR.In effect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments-further diversifying the potential hybridization sitesduring the annealing step of the reaction.

[1325] A variety of reaction conditions could be utilized to carry-outthe DNA shuffling reaction. However, specific reaction conditions forDNA shuffling are provided, for example, in PNAS, 91:10747, (1994).Briefly:

[1326] Prepare the DNA substrate to be subjected to the DNA shufflingreaction. Preparation may be in the form of simply purifying the DNAfrom contaminating cellular material, chemicals, buffers,oligonucleotide primers, deoxynucleotides, RNAs, etc., and may entailthe use of DNA purification kits as those provided by Qiagen, Inc., orby the Promega, Corp., for example.

[1327] Once the DNA substrate has been purified, it would be subjectedto Dnase I digestion. About 2-4 ug of the DNA substrate(s) would bedigested with .0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mMTris-HCL, pH 7.4/1 mM MgCl2 for 10-20 min. at room temperature. Theresulting fragments of 10-50 bp could then be purified by running themthrough a 2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

[1328] The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

[1329] The resulting shuffled, assembled, and amplified product can bepurified using methods well known in the art (e.g., Qiagen PCRpurification kits) and then subsequently cloned using appropriaterestriction enzymes.

[1330] Although a number of variations of DNA shuffling have beenpublished to date, such variations would be obvious to the skilledartisan and are encompassed by the invention. The DNA shuffling methodcan also be tailored to the desired level of mutagenesis using themethods described by Zhao, et al. (Nucl Acid Res., 25(6): 1307-1308,(1997).

[1331] As described above, once the randomized pool has been created, itcan then be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

[1332] DNA shuffling has several advantages. First, it makes use ofbeneficial mutations. When combined with screening, DNA shuffling allowsthe discovery of the best mutational combinations and does not assumethat the best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

[1333] A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

[1334] Finally, recombination enables parallel processing. Thisrepresents a significant advantage since there are likely multiplecharacteristics that would make a protein more desirable (e.g.solubility, activity, etc.). Since it is increasingly difficult toscreen for more than one desirable trait at a time, other methods ofmolecular evolution tend to be inhibitory. However, using recombination,it would be possible to combine the randomized fragments of the bestrepresentative variants for the various traits, and then select formultiple properties at once.

[1335] DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

[1336] Likewise, the invention encompasses the application of DNAshuffling technology to the evolution of polynucleotides andpolypeptides of the invention, wherein one or more cycles of DNAshuffling include, in addition to the gene template DNA,oligonucleotides coding for known allelic sequences, optimized codonsequences, known variant sequences, known polynucleotide polymorphismsequences, known ortholog sequences, known homologue sequences,additional homologous sequences, additional non-homologous sequences,sequences from another species, and any number and combination of theabove.

[1337] In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

[1338] Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 26 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

[1339] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:1. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70degrees C., using buffer solutions described in Sidransky et al.,Science 252:706 (1991).

[1340] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

[1341] PCR products are cloned into T-tailed vectors as described inHolton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced withT7 polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

[1342] Genomic rearrangements are also observed as a method ofdetermining alterations in a gene corresponding to a polynucleotide.Genomic clones isolated according to Example 15 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

[1343] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 27 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

[1344] A polypeptide of the present invention can be detected in abiological sample, and if an increased or decreased level of thepolypeptide is detected, this polypeptide is a marker for a particularphenotype. Methods of detection are numerous, and thus, it is understoodthat one skilled in the art can modify the following assay to fit theirparticular needs.

[1345] For example, antibody-sandwich ELISAs are used to detectpolypeptides in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies, at a finalconcentration of 0.2 to 10 ug/ml. The antibodies are either monoclonalor polyclonal and are produced by the method described elsewhere herein.The wells are blocked so that non-specific binding of the polypeptide tothe well is reduced.

[1346] The coated wells are then incubated for >2 hours at RT with asample containing the polypeptide. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedpolypeptide.

[1347] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[1348] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot polypeptide concentration on theX-axis (log scale) and fluorescence or absorbance of the Y-axis (linearscale). Interpolate the concentration of the polypeptide in the sampleusing the standard curve.

Example 28 Formulation

[1349] The invention also provides methods of treatment and/orprevention diseases, disorders, and/or conditions (such as, for example,any one or more of the diseases or disorders disclosed herein) byadministration to a subject of an effective amount of a Therapeutic. Bytherapeutic is meant a polynucleotides or polypeptides of the invention(including fragments and variants), agonists or antagonists thereof,and/or antibodies thereto, in combination with a pharmaceuticallyacceptable carrier type (e.g., a sterile carrier).

[1350] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[1351] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[1352] Therapeutics can be administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1353] In yet an additional embodiment, the Therapeutics of theinvention are delivered orally using the drug delivery technologydescribed in U.S. Pat. 6,258,789, which is hereby incorporated byreference herein.

[1354] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[1355] Therapeutics of the invention may also be suitably administeredby sustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[1356] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res.15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988).

[1357] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: U.S. DE Pat. No.3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal Therapeutic.

[1358] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[1359] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[1360] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[1361] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[1362] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[1363] The Therapeutic will typically be formulated in such vehicles ata concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml,at a pH of about 3 to 8. It will be understood that the use of certainof the foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[1364] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[1365] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[1366] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[1367] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B. haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[1368] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[1369] In one embodiment, the Therapeutics of the invention areadministered in combination with members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with theTherapeutics of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[1370] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR(zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT(stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE (nevirapine),RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR(ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfinavir). In aspecific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

[1371] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE,ISONIAZID, RIFAMPIN, PYRAZINAMIDE, ETHAMBUTOL, RIFABUTIN,CLARITHROMYCIN, AZITHROMYCIN, GANCICLOVIR, FOSCARNET, CIDOFOVIR,FLUCONAZOLE, ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR,PYRIMETHAMINE, LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONEto prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONIAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylacticallytreat or prevent an opportunistic Mycobacterium avium complex infection.In another specific embodiment, Therapeutics of the invention are usedin any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCINto prophylactically treat or prevent an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, Therapeutics ofthe invention are used in any combination with GANCICLOVIR, FOSCARNET,and/or CIDOFOVIR to prophylactically treat or prevent an opportunisticcytomegalovirus infection. In another specific embodiment, Therapeuticsof the invention are used in any combination with FLUCONAZOLE,ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or preventan opportunistic fungal infection. In another specific embodiment,Therapeutics of the invention are used in any combination with ACYCLOVIRand/or FAMCICOLVIR to prophylactically treat or prevent an opportunisticherpes simplex virus type I and/or type II infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with PYRIMETHAMINE and/or LEUCOVORIN to prophylacticallytreat or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with LEUCOVORIN and/or NEUPOGEN to prophylacticallytreat or prevent an opportunistic bacterial infection.

[1372] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[1373] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

[1374] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[1375] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE (OKT3),SANDIMMUNE/NEORAL/SANGDYA (cyclosporin), PROGRAF (tacrolimus), CELLCEPT(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE(sirolimus). In a specific embodiment, immunosuppressants may be used toprevent rejection of organ or bone marrow transplantation.

[1376] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE.In a specific embodiment, Therapeutics of the invention are administeredin combination with intravenous immune globulin preparations intransplantation therapy (e.g., bone marrow transplant).

[1377] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[1378] In another embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[1379] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[1380] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

[1381] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[1382] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE(SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).

[1383] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[1384] In an additional embodiment, the Therapeutics of the inventionare administered in combination with other immune factors. Immunefactors that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Ly9, CD2, CD48, CD58, 2B4, CD84,CDw150, CTLA4, CTLA4Ig, Bsl1, Bsl2, Bsl3, BLYS, TRAIL, APRIL, B7, B7antagonists, B7 agonists, and Ret16.

[1385] In a specific embodiment, formulations of the present inventionmay further comprise antagonists of P-glycoprotein (also referred to asthe multiresistance protein, or PGP), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). P-glycoprotein is well known for decreasingthe efficacy of various drug administrations due to its ability toexport intracellular levels of absorbed drug to the cell exterior. Whilethis activity has been particularly pronounced in cancer cells inresponse to the administration of chemotherapy regimens, a variety ofother cell types and the administration of other drug classes have beennoted (e.g., T-cells and anti-HIV drugs). In fact, certain mutations inthe PGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each parent—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

[1386] Moreover, in another specific embodiment, formulations of thepresent invention may further comprise antagonists of OATP2 (alsoreferred to as the multiresistance protein, or MRP2), includingantagonists of its encoding polynucleotides (e.g., antisenseoligonucleotides, ribozymes, zinc-finger proteins, etc.). The inventionalso further comprises any additional antagonists known to inhibitproteins thought to be attributable to a multidrug resistant phenotypein proliferating cells.

[1387] Preferred antagonists that formulations of the present maycomprise include the potent P-glycoprotein inhibitor elacridar, and/orLY-335979. Other P-glycoprotein inhibitors known in the art are alsoencompassed by the present invention.

[1388] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 29 Method of Treating Decreased Levels of the Polypeptide

[1389] The present invention relates to a method for treating anindividual in need of an increased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an agonistof the invention (including polypeptides of the invention). Moreover, itwill be appreciated that conditions caused by a decrease in the standardor normal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

[1390] For example, a patient with decreased levels of a polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided herein.

Example 30 Method of Treating Increased Levels of the Polypeptide

[1391] The present invention also relates to a method of treating anindividual in need of a decreased level of a polypeptide of theinvention in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of anantagonist of the invention (including polypeptides and antibodies ofthe invention).

[1392] In one example, antisense technology is used to inhibitproduction of a polypeptide of the present invention. This technology isone example of a method of decreasing levels of a polypeptide,preferably a secreted form, due to a variety of etiologies, such ascancer. For example, a patient diagnosed with abnormally increasedlevels of a polypeptide is administered intravenously antisensepolynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days.This treatment is repeated after a 7-day rest period if the treatmentwas well tolerated. The formulation of the antisense polynucleotide isprovided herein.

Example 31 Method of Treatment Using Gene Therapy—Ex Vivo

[1393] One method of gene therapy transplants fibroblasts, which arecapable of expressing a polypeptide, onto a patient. Generally,fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[1394] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[1395] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[1396] The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 15 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

[1397] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[1398] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether protein is produced.

[1399] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 32 Gene Therapy Using Endogenous Genes Corresponding toPolynucleotides of the Invention

[1400] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. NO: 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

[1401] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[1402] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[1403] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[1404] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous polynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

[1405] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

[1406] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the locus correspondingto the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end.Two non-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′ end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′ end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

[1407] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×106cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[1408] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

[1409] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 33 Method of Treatment Using Gene Therapy—In Vivo

[1410] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques and methods areknown in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat.No. 5693622, 5705151, 5580859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

[1411] The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[1412] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. N Y Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

[1413] The polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[1414] The polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

[1415] For the naked polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[1416] The dose response effects of injected polynucleotide in muscle invivo is determined as follows. Suitable template DNA for production ofmRNA coding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

[1417] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The template DNA is injected in 0.1 ml of carrierin a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[1418] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15um cross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 34 Transgenic Animals

[1419] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[1420] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[1421] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[1422] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[1423] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[1424] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[1425] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of polypeptides of the present invention, studyingdiseases, disorders, and/or conditions associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch diseases, disorders, and/or conditions.

Example 35 Knock-out Animals

[1426] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

[1427] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[1428] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[1429] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[1430] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 36 Method of Isolating Antibody Fragments Directed AgainstLTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, or LTRPC3l from a Libraryof scFvs

[1431] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, orLTRPC3l to which the donor may or may not have been exposed (see e.g.,U.S. Pat. 5,885,793 incorporated herein by reference in its entirety).

[1432] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in PCT publication WO 92/01047. To rescuephage displaying antibody fragments, approximately 109 E. coli harboringthe phagemid are used to inoculate 50 ml of 2×TY containing 1% glucoseand 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8with shaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

[1433] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

[1434] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[1435] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scFvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

[1436] Moreover, in another preferred method, the antibodies directedagainst the polypeptides of the present invention may be produced inplants. Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 37 Identification and Cloning of VH and VL Domains of AntibodiesDirected Against the LTRPC3g, LTRPC3h, LTRPC3i, LTRPC3j, LTRPC3k, orLTRPC3l Polypeptide

[1437] VH and VL domains may be identified and cloned from cell linesexpressing an antibody directed against a LTRPC3g, LTRPC3h, LTRPC3i,LTRPC3j, LTRPC3k, or LTRPC3l epitope by performing PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may be lysed using the TRIzolreagent (Life Technologies, Rockville, Md.) and extracted with one fifthvolume of chloroform. After addition of chloroform, the solution isallowed to incubate at room temperature for 10 minutes, and thencentrifuged at 14, 000 rpm for 15 minutes at 4 C in a tabletopcentrifuge. The supernatant is collected and RNA is precipitated usingan equal volume of isopropanol. Precipitated RNA is pelleted bycentrifuging at 14, 000 rpm for 15 minutes at 4 C in a tabletopcentrifuge.

[1438] Following centrifugation, the supernatant is discarded and washedwith 75% ethanol. Following the wash step, the RNA is centrifuged againat 800 rpm for 5 minutes at 4 C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 60 C for 10 minutes. Quantities of RNA can be determined usingoptical density measurements. cDNA may be synthesized, according tomethods well-known in the art and/or described herein, from 1.5-2. 5micrograms of RNA using reverse transciptase and random hexamer primers.cDNA is then used as a template for PCR amplification of VH and VLdomains.

[1439] Primers used to amplify VH and VL genes are shown below.Typically a PCR reaction makes use of a single 5′ primer and a single 3′primer. Sometimes, when the amount of available RNA template islimiting, or for greater efficiency, groups of 5′ and/or 3′ primers maybe used. For example, sometimes all five VH-5′ primers and all JH3′primers are used in a single PCR reaction. The PCR reaction is carriedout in a 50 microliter volume containing 1×PCR buffer, 2 mM of eachdNTP, 0.7 units of High Fidelity Taq polymerse, 5′ primer mix, 3′ primermix and 7. 5 microliters of cDNA. The 5′ and 3′ primer mix of both VHand VL can be made by pooling together 22 pmole and 28 pmole,respectively, of each of the individual primers. PCR conditions are: 96C for 5 minutes; followed by 25 cycles of 94 C for 1 minute, 50 C for 1minute, and 72 C for 1 minute; followed by an extension cycle of 72 Cfor 10 minutes. After the reaction has been completed, sample tubes maybe stored at 4 C. Primer Sequences Used to Amplify VH domains Primername Primer Sequence SEQ ID NO: Hu VH1-5′ CAGGTGCAGCTGGTGCAGTCTGG 132 HuVH2-5′ CAGGTCAACTTAAGGGAGTCTGG 133 Hu VH3-5′ GAGGTGCAGCTGGTGGAGTCTGG 134Hu VH4-5′ CAGGTGCAGCTGCAGGAGTCGGG 135 Hu VH5-5′ GAGGTGCAGCTGTTGCAGTCTGC136 Hu VH6-5′ CAGGTACAGCTGCAGCAGTCAGG 137 Hu JH1-5′TGAGGAGACGGTGACCAGGGTGCC 138 Hu JH3-5′ TGAAGAGACGGTGACCATTGTCCC 139 HuJH4-5′ TGAGGAGACGGTGACCAGGGTTCC 140 Hu JH6-5′ TGAGGAGACGGTGACCGTGGTCCC141

[1440] Primer Sequences Used to Amplify VL domains SEQ Primer namePrimer Sequence ID NO: Hu Vkappa1-5′ GACATCCAGATGACCCAGTCTCC 142 HuVkappa2a-5′ GATGTTGTGATGACTCAGTCTCC 143 Hu Vkappa2b-5′GATATTGTGATGACTCAGTCTCC 144 Hu Vkappa3-5′ GAAATTGTGTTGACGCAGTCTCC 145 HuVkappa4-5′ GACATCGTGATGACCCAGTCTCC 146 Hu Vkappa5-5′GAAACGACACTCACGCAGTCTCC 147 Hu Vkappa6-5′ GAAATTGTGCTGACTCAGTCTCC 148 HuVlambda1-5′ CAGTCTGTGTTGACGCAGCCGCC 149 Hu Vlambda2-5′CAGTCTGCCCTGACTCAGCCTGC 150 Hu Vlambda3-5′ TCCTATGTGCTGACTCAGCCACC 151Hu Vlambda3b-5′ TCTTCTGAGCTGACTCAGGACCC 152 Hu Vlambda4-5′CACGTTATACTGACTCAACCGCC 153 Hu Vlambda5-5′ CAGGCTGTGCTCACTCAGCCGTC 154Hu Vlambda6-5′ AATTTTATGCTGACTCAGCCCCA 155 Hu Jkappa1-3′ACGTTTGATTTCCACCTTGGTCCC 156 Hu Jkappa2-3′ ACGTTTGATCTCCAGCTTGGTCCC 157Hu Jkappa3-3′ ACGTTTGATATCCACTTTGGTCCC 158 Hu Jkappa4-3′ACGTTTGATCTCCACCTTGGTCCC 159 Hu Jkappa5-3′ ACGTTTAATCTCCAGTCGTGTCCC 160Hu Vlambda1-3′ CAGTCTGTGTTGACGCAGCCGCC 161 Hu Vlambda2-3′CAGTCTGCCCTGACTCAGCCTGC 162 Hu Vlambda3-3′ TCCTATGTGCTGACTCAGCCACC 163Hu Vlambda3b-3′ TCTTCTGAGCTGACTCAGGACCC 164 Hu Vlambda4-3′CACGTTATACTGACTCAACCGCC 165 Hu Vlambda5-3′ CAGGCTGTGCTCACTCAGCCGTC 166Hu Vlambda6-3′ AATTTTATGCTGACTCAGCCCCA 167

[1441] PCR samples are then electrophoresed on a 1.3% agarose gel. DNAbands of the expected sizes (-506 base pairs for VH domains, and 344base pairs for VL domains) can be cut out of the gel and purified usingmethods well known in the art and/or described herein.

[1442] Purified PCR products can be ligated into a PCR cloning vector(TA vector from Invitrogen Inc., Carlsbad, Calif.). Individual clonedPCR products can be isolated after transfection of E. coli andblue/white color selection. Cloned PCR products may then be sequencedusing methods commonly known in the art and/or described herein.

[1443] The PCR bands containing the VH domain and the VL domains canalso be used to create full-length Ig expression vectors. VH and VLdomains can be cloned into vectors containing the nucleotide sequencesof a heavy (e. g., human IgG1 or human IgG4) or light chain (human kappaor human ambda) constant regions such that a complete heavy or lightchain molecule could be expressed from these vectors when transfectedinto an appropriate host cell. Further, when cloned heavy and lightchains are both expressed in one cell line (from either one or twovectors), they can assemble into a complete functional antibody moleculethat is secreted into the cell culture medium. Methods usingpolynucleotides encoding VH and VL antibody domain to generateexpression vectors that encode complete antibody molecules are wellknown within the art.

Example 38 Biological Effects of Polypeptides of the Invention Astrocyteand Neuronal Assays

[1444] Recombinant polypeptides of the invention, expressed inEscherichia coli and purified as described above, can be tested foractivity in promoting the survival, neurite outgrowth, or phenotypicdifferentiation of cortical neuronal cells and for inducing theproliferation of glial fibrillary acidic protein immunopositive cells,astrocytes. The selection of cortical cells for the bioassay is based onthe prevalent expression of FGF-1 and FGF-2 in cortical structures andon the previously reported enhancement of cortical neuronal survivalresulting from FGF-2 treatment. A thymidine incorporation assay, forexample, can be used to elucidate a polypeptide of the invention'sactivity on these cells.

[1445] Moreover, previous reports describing the biological effects ofFGF-2 (basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays

[1446] Human lung fibroblasts are obtained from Clonetics (San Diego,Calif.) and maintained in growth media from Clonetics. Dermalmicrovascular endothelial cells are obtained from Cell Applications (SanDiego, Calif.). For proliferation assays, the human lung fibroblasts anddermal microvascular endothelial cells can be cultured at 5,000cells/well in a 96-well plate for one day in growth medium. The cellsare then incubated for one day in 0.1% BSA basal medium. After replacingthe medium with fresh 0.1% BSA medium, the cells are incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE2 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or polypeptides of the invention with or withoutIL-1(for 24 hours. The supernatants are collected and assayed for PGE2by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the humanlung fibroblasts are cultured at 5,000 cells/well in a 96-well plate forone day. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1(for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, MA).

[1447] Human lung fibroblasts are cultured with FGF-2 or polypeptides ofthe invention for 3 days in basal medium before the addition of AlamarBlue to assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/mi which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models

[1448] The loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP+ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

[1449] It has been demonstrated in tissue culture paradigms that FGF-2(basic FGF) has trophic activity towards nigral dopaminergic neurons(Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

[1450] Based on the data with FGF-2, polypeptides of the invention canbe evaluated to determine whether it has an action similar to that ofFGF-2 in enhancing dopaminergic neuronal survival in vitro and it canalso be tested in vivo for protection of dopaminergic neurons in thestriatum from the damage associated with MPTP treatment. The potentialeffect of a polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (Ni). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

[1451] Since the dopaminergic neurons are isolated from animals atgestation day 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

[1452] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 39 The Effect of Polypeptides of the Invention on the Growth ofVascular Endothelial Cells

[1453] On day 1, human umbilical vein endothelial cells (HUVEC) areseeded at 2-5×104 cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium is replaced with M199 containing 10% FBS, 8 units/mlheparin. A polypeptide having the amino acid sequence of SEQ ID NO:2,and positive controls, such as VEGF and basic FGF (bFGF) are added, atvarying concentrations. On days 4 and 6, the medium is replaced. On day8, cell number is determined with a Coulter Counter.

[1454] An increase in the number of HUVEC cells indicates that thepolypeptide of the invention may proliferate vascular endothelial cells.

[1455] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 40 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

[1456] For evaluation of mitogenic activity of growth factors, thecolorimetric MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

[1457] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 41 Suppression of TNF Alpha-induced Adhesion Molecule Expressionby a Polypeptide of the Invention

[1458] The recruitment of lymphocytes to areas of inflammation andangiogenesis involves specific receptor-ligand interactions between cellsurface adhesion molecules (CAMs) on lymphocytes and the vascularendothelium. The adhesion process, in both normal and pathologicalsettings, follows a multi-step cascade that involves intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin)expression on endothelial cells (EC). The expression of these moleculesand others on the vascular endothelium determines the efficiency withwhich leukocytes may adhere to the local vasculature and extravasateinto the local tissue during the development of an inflammatoryresponse. The local concentration of cytokines and growth factorparticipate in the modulation of the expression of these CAMs.

[1459] Tumor necrosis factor alpha (TNF-a), a potent proinflammatorycytokine, is a stimulator of all three CAMs on endothelial cells and maybe involved in a wide variety of inflammatory responses, often resultingin a pathological outcome.

[1460] The potential of a polypeptide of the invention to mediate asuppression of TNF-a induced CAM expression can be examined. A modifiedELISA assay which uses ECs as a solid phase absorbent is employed tomeasure the amount of CAM expression on TNF-a treated ECs whenco-stimulated with a member of the FGF family of proteins.

[1461] To perform the experiment, human umbilical vein endothelial cell(HUVEC) cultures are obtained from pooled cord harvests and maintainedin growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidifiedincubator containing 5% CO2. HUVECs are seeded in 96-well plates atconcentrations of 1×104 cells/well in EGM medium at 37 degree C. for18-24 hrs or until confluent. The monolayers are subsequently washed 3times with a serum-free solution of RPMI-1640 supplemented with 100 U/mlpenicillin and 100 mg/ml streptomycin, and treated with a given cytokineand/or growth factor(s) for 24 h at 37 degree C. Following incubation,the cells are then evaluated for CAM expression.

[1462] Human Umbilical Vein Endothelial cells (HUVECs) are grown in astandard 96 well plate to confluence. Growth medium is removed from thecells and replaced with 90 ul of 199 Medium (10% FBS). Samples fortesting and positive or negative controls are added to the plate intriplicate (in 10 ul volumes). Plates are incubated at 37 degree C. foreither 5 h (selectin and integrin expression) or 24 h (integrinexpression only). Plates are aspirated to remove medium and. 100 μl of0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well.Plates are held at 4° C. for 30 min.

[1463] Fixative is then removed from the wells and wells are washed 1×with PBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry.Add 10 μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

[1464] Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase(1:5,000 dilution) to each well and incubated at 37° C. for 30 min.Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-NitrophenolPhosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μlof pNPP substrate in glycine buffer is added to each test well. Standardwells in triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000(100) >10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicatewells and the resulting AP content in each well is 5.50 ng, 1.74 ng,0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each ofthe standard wells. The plate must be incubated at 37° C. for 4h. Avolume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

[1465] One skilled in the art could easily modify the exemplifiedstudies to test the activity of polynucleotides of the invention (e.g.,gene therapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

[1466] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[1467] The entire disclosure of each document cited (including patents,patent applications, journal articles, abstracts, laboratory manuals,books, or other disclosures) in the Background of the Invention,Detailed Description, and Examples is hereby incorporated herein byreference. Further, the hard copy of the sequence listing submittedherewith and the corresponding computer readable form are bothincorporated herein by reference in their entireties.

1 171 1 5201 DNA Homo sapiens CDS (72)..(5198) 1 cgcccgcggc gaggagccagcgagagcgct cggcgctggg ctgtttcccg gccgagggag 60 gcgaacttct c atg ggg aagaag tgg agg gat gcg gcg gaa atg gag cgg 110 Met Gly Lys Lys Trp Arg AspAla Ala Glu Met Glu Arg 1 5 10 ggc tgc tcc gac cgc gag gac aac gcg gagagc cgc aga cgc agc cgg 158 Gly Cys Ser Asp Arg Glu Asp Asn Ala Glu SerArg Arg Arg Ser Arg 15 20 25 agc gcc agc cgg ggc agg ttt gcc gag tcg tggaaa agg tta agt tcc 206 Ser Ala Ser Arg Gly Arg Phe Ala Glu Ser Trp LysArg Leu Ser Ser 30 35 40 45 aag cag ggg tcc acc aaa cgc tcg gga ctc ccgtcg cag cag acg ccg 254 Lys Gln Gly Ser Thr Lys Arg Ser Gly Leu Pro SerGln Gln Thr Pro 50 55 60 gct cag aaa tcc tgg ata gaa aga gca ttt tat aaaaga gaa tgt gtc 302 Ala Gln Lys Ser Trp Ile Glu Arg Ala Phe Tyr Lys ArgGlu Cys Val 65 70 75 cac atc ata ccc agc acc aaa gac ccc cat agg tgt tgctgt ggg cgt 350 His Ile Ile Pro Ser Thr Lys Asp Pro His Arg Cys Cys CysGly Arg 80 85 90 ctg ata ggc cag cat gtt ggc ctc acc ccc agt atc tcc gtgctt cag 398 Leu Ile Gly Gln His Val Gly Leu Thr Pro Ser Ile Ser Val LeuGln 95 100 105 aat gag aaa aat gaa agt cgc ctc tcc cga aat gac atc cagtct gaa 446 Asn Glu Lys Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln SerGlu 110 115 120 125 aag tgg tcc atc agc aaa cac act caa ctc agc cct acggat gct ttt 494 Lys Trp Ser Ile Ser Lys His Thr Gln Leu Ser Pro Thr AspAla Phe 130 135 140 ggg acc att gag ttc caa gga ggt ggc cat tcc aac aaagcc atg tat 542 Gly Thr Ile Glu Phe Gln Gly Gly Gly His Ser Asn Lys AlaMet Tyr 145 150 155 gtg cga gta tct ttt gat aca aaa cct gat ctc ctc ttacac ctg atg 590 Val Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu HisLeu Met 160 165 170 acc aag gaa tgg cag ttg gag ctt ccc aag ctt ctc atctct gtc cat 638 Thr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile SerVal His 175 180 185 ggg ggc ctg cag aac ttt gaa ctc cag cca aaa ctc aagcaa gtc ttt 686 Gly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys GlnVal Phe 190 195 200 205 ggg aaa ggg ctc atc aaa gca gca atg aca act ggagcg tgg ata ttc 734 Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly AlaTrp Ile Phe 210 215 220 act gga ggg gtt aac aca ggt gtt att cgt cat gttggc gat gcc ttg 782 Thr Gly Gly Val Asn Thr Gly Val Ile Arg His Val GlyAsp Ala Leu 225 230 235 aag gat cat gcc tct aag tct cga gga aag ata tgcacc ata ggt att 830 Lys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys ThrIle Gly Ile 240 245 250 gcc ccc tgg gga att gtg gaa aac cag gag gac ctcatt gga aga gat 878 Ala Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu IleGly Arg Asp 255 260 265 gtt gtc cgg cca tac cag acc atg tcc aat ccc atgagc aag ctc act 926 Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met SerLys Leu Thr 270 275 280 285 gtt ctc aac agc atg cat tcc cac ttc att ctggct gac aac ggg acc 974 Val Leu Asn Ser Met His Ser His Phe Ile Leu AlaAsp Asn Gly Thr 290 295 300 act gga aaa tat gga gca gag gtg aaa ctt cgaaga caa ctg gaa aag 1022 Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg ArgGln Leu Glu Lys 305 310 315 cat att tca ctc cag aag ata aac aca aga atcggt caa ggt gtt cct 1070 His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile GlyGln Gly Val Pro 320 325 330 gtg gtg gca ctc ata gtg gaa gga gga ccc aatgtg atc tcg att gtt 1118 Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn ValIle Ser Ile Val 335 340 345 ttg gag tac ctt cga gac acc cct ccc gtg ccagtg gtt gtc tgt gat 1166 Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro ValVal Val Cys Asp 350 355 360 365 ggg agt gga cgg gca tcg gac atc ctg gccttt ggg cat aaa tac tca 1214 Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala PheGly His Lys Tyr Ser 370 375 380 gaa gaa ggc gga ctg ata aat gaa tct ttgagg gac cag ctg ttg gtg 1262 Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu ArgAsp Gln Leu Leu Val 385 390 395 act ata cag aag act ttc aca tac act cgaacc caa gct cag cat ctg 1310 Thr Ile Gln Lys Thr Phe Thr Tyr Thr Arg ThrGln Ala Gln His Leu 400 405 410 ttc atc atc ctc atg gag tgc atg aag aagaag gaa ttg att acg gta 1358 Phe Ile Ile Leu Met Glu Cys Met Lys Lys LysGlu Leu Ile Thr Val 415 420 425 ttt cgg atg gga tca gaa gga cac cag gacatt gat ttg gct atc ctg 1406 Phe Arg Met Gly Ser Glu Gly His Gln Asp IleAsp Leu Ala Ile Leu 430 435 440 445 aca gct tta ctc aaa gga gcc aat gcctcg gcc cca gac caa ctg agc 1454 Thr Ala Leu Leu Lys Gly Ala Asn Ala SerAla Pro Asp Gln Leu Ser 450 455 460 tta gct tta gcc tgg aac aga gtc gacatc gct cgc agc cag atc ttt 1502 Leu Ala Leu Ala Trp Asn Arg Val Asp IleAla Arg Ser Gln Ile Phe 465 470 475 att tac ggg caa cag tgg ccg gtg ggatct ctg gag caa gcc atg ttg 1550 Ile Tyr Gly Gln Gln Trp Pro Val Gly SerLeu Glu Gln Ala Met Leu 480 485 490 gat gcc tta gtt ctg gac aga gtg gatttt gtg aaa tta ctc ata gag 1598 Asp Ala Leu Val Leu Asp Arg Val Asp PheVal Lys Leu Leu Ile Glu 495 500 505 aat gga gta agc atg cac cgt ttt ctcacc atc tcc aga cta gag gaa 1646 Asn Gly Val Ser Met His Arg Phe Leu ThrIle Ser Arg Leu Glu Glu 510 515 520 525 ttg tac aat acg aga cat ggg ccctca aat aca ttg tac cac ttg gtc 1694 Leu Tyr Asn Thr Arg His Gly Pro SerAsn Thr Leu Tyr His Leu Val 530 535 540 agg gat gtc aaa aag ggg aac ctgccc cca gac tac aga atc agc ctg 1742 Arg Asp Val Lys Lys Gly Asn Leu ProPro Asp Tyr Arg Ile Ser Leu 545 550 555 att gac atc ggc ctg gtg atc gagtac ctg atg ggc ggg gct tat cgc 1790 Ile Asp Ile Gly Leu Val Ile Glu TyrLeu Met Gly Gly Ala Tyr Arg 560 565 570 tgc aac tac acg cgc aag cgc ttccgg acc ctc tac cac aac ctc ttc 1838 Cys Asn Tyr Thr Arg Lys Arg Phe ArgThr Leu Tyr His Asn Leu Phe 575 580 585 ggc ccc aag agg ccc aaa gcc ttgaaa ctg ctg gga atg gag gat gat 1886 Gly Pro Lys Arg Pro Lys Ala Leu LysLeu Leu Gly Met Glu Asp Asp 590 595 600 605 att ccc ttg agg cga gga agaaag aca acc aag aaa cgt gaa gaa gag 1934 Ile Pro Leu Arg Arg Gly Arg LysThr Thr Lys Lys Arg Glu Glu Glu 610 615 620 gtg gac att gac ttg gat gatcct gag atc aac cac ttc ccc ttc cct 1982 Val Asp Ile Asp Leu Asp Asp ProGlu Ile Asn His Phe Pro Phe Pro 625 630 635 ttc cat gag ctc atg gtg tgggct gtt ctc atg aag cgg cag aag atg 2030 Phe His Glu Leu Met Val Trp AlaVal Leu Met Lys Arg Gln Lys Met 640 645 650 gcc ctg ttc ttc tgg cag cacggt gag gag gcc atg gcc aag gcc ctg 2078 Ala Leu Phe Phe Trp Gln His GlyGlu Glu Ala Met Ala Lys Ala Leu 655 660 665 gtg gcc tgc aag ctc tgc aaagcc atg gct cat gag gcc tct gag aac 2126 Val Ala Cys Lys Leu Cys Lys AlaMet Ala His Glu Ala Ser Glu Asn 670 675 680 685 gac atg gtt gac gac atttcc cag gag ctg aat cac aat tcc aga gac 2174 Asp Met Val Asp Asp Ile SerGln Glu Leu Asn His Asn Ser Arg Asp 690 695 700 ttt ggc cag ctg gct gtggag ctc ctg gac cag tcc tac aag cag gac 2222 Phe Gly Gln Leu Ala Val GluLeu Leu Asp Gln Ser Tyr Lys Gln Asp 705 710 715 gaa cag ctg gcc atg aaactg ctg acg tat gag ctg aag aac tgg agc 2270 Glu Gln Leu Ala Met Lys LeuLeu Thr Tyr Glu Leu Lys Asn Trp Ser 720 725 730 aac gcc acg tgc ctg cagctt gcc gtg gct gcc aaa cac cgc gac ttc 2318 Asn Ala Thr Cys Leu Gln LeuAla Val Ala Ala Lys His Arg Asp Phe 735 740 745 atc gcg cac acg tgc agccag atg ctg ctc acc gac atg tgg atg ggc 2366 Ile Ala His Thr Cys Ser GlnMet Leu Leu Thr Asp Met Trp Met Gly 750 755 760 765 cgg ctc cgc atg cgcaag aac tca ggc ctc aag gta att ctg gga att 2414 Arg Leu Arg Met Arg LysAsn Ser Gly Leu Lys Val Ile Leu Gly Ile 770 775 780 cta ctt cct cct tcaatt ctc agc ttg gag ttc aag aac aaa gac gac 2462 Leu Leu Pro Pro Ser IleLeu Ser Leu Glu Phe Lys Asn Lys Asp Asp 785 790 795 atg ccc tat atg tctcag gcc cag gaa atc cac ctc caa gag aag gag 2510 Met Pro Tyr Met Ser GlnAla Gln Glu Ile His Leu Gln Glu Lys Glu 800 805 810 gca gaa gaa cca gagaag ccc aca aag gaa aaa gag gaa gag gac atg 2558 Ala Glu Glu Pro Glu LysPro Thr Lys Glu Lys Glu Glu Glu Asp Met 815 820 825 gag ctc aca gca atgttg gga cga aac aac ggg gag tcc tcc agg aag 2606 Glu Leu Thr Ala Met LeuGly Arg Asn Asn Gly Glu Ser Ser Arg Lys 830 835 840 845 aag gat gaa gaggaa gtt cag agc aag cac cgg tta atc ccc ctc ggc 2654 Lys Asp Glu Glu GluVal Gln Ser Lys His Arg Leu Ile Pro Leu Gly 850 855 860 aga aaa atc tatgaa ttc tac aat gca ccc atc gtg aag ttc tgg ttc 2702 Arg Lys Ile Tyr GluPhe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe 865 870 875 tac aca ctg gcgtat atc gga tac ctg atg ctc ttc aac tat atc gtg 2750 Tyr Thr Leu Ala TyrIle Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val 880 885 890 tta gtg aag atggaa cgc tgg ccg tcc acc cag gaa tgg atc gta atc 2798 Leu Val Lys Met GluArg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile 895 900 905 tcc tat att ttcacc ctg gga ata gaa aag atg aga gag att ctg atg 2846 Ser Tyr Ile Phe ThrLeu Gly Ile Glu Lys Met Arg Glu Ile Leu Met 910 915 920 925 tca gag ccaggg aag ttg cta cag aaa gtg aag gta tgg ctg cag gag 2894 Ser Glu Pro GlyLys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu 930 935 940 tac tgg aatgtc acg gac ctc atc gcc atc ctt ctg ttt tct gtc gga 2942 Tyr Trp Asn ValThr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly 945 950 955 atg atc cttcgt ctc caa gac cag ccc ttc agg agt gac ggg agg gtc 2990 Met Ile Leu ArgLeu Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val 960 965 970 atc tac tgcgtg aac atc att tac tgg tat atc cgt ctc cta gac atc 3038 Ile Tyr Cys ValAsn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile 975 980 985 ttc ggc gtgaac aag tat ttg ggc ccg tat gta atg atg att gga aaa 3086 Phe Gly Val AsnLys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys 990 995 1000 1005 atgatg ata gac atg atg tac ttt gtc atc att atg ctg gtg gtt 3131 Met Met IleAsp Met Met Tyr Phe Val Ile Ile Met Leu Val Val 1010 1015 1020 ctg atgagc ttt ggg gtc gcc agg caa gcc atc ctt ttt ccc aat 3176 Leu Met Ser PheGly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn 1025 1030 1035 gag gag ccatca tgg aaa ctg gcc aag aac atc ttc tac atg ccc 3221 Glu Glu Pro Ser TrpLys Leu Ala Lys Asn Ile Phe Tyr Met Pro 1040 1045 1050 tat tgg atg atttat ggg gaa gtg ttt gcg gac cag ata gac cct 3266 Tyr Trp Met Ile Tyr GlyGlu Val Phe Ala Asp Gln Ile Asp Pro 1055 1060 1065 ccc tgt gga cag aatgag acc cga gag gat ggt aaa ata atc cag 3311 Pro Cys Gly Gln Asn Glu ThrArg Glu Asp Gly Lys Ile Ile Gln 1070 1075 1080 ctg cct ccc tgc aag acagga gct tgg atc gtg ccg gcc atc atg 3356 Leu Pro Pro Cys Lys Thr Gly AlaTrp Ile Val Pro Ala Ile Met 1085 1090 1095 gcc tgc tac ctc tta gtg gcaaac atc ttg ctg gtc aac ctc ctc 3401 Ala Cys Tyr Leu Leu Val Ala Asn IleLeu Leu Val Asn Leu Leu 1100 1105 1110 att gct gtc ttt aac aat aca tttttt gaa gta aaa tcg ata tcc 3446 Ile Ala Val Phe Asn Asn Thr Phe Phe GluVal Lys Ser Ile Ser 1115 1120 1125 aac caa gtc tgg aag ttt cag agg tatcag ctc atc atg act ttc 3491 Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln LeuIle Met Thr Phe 1130 1135 1140 cat gaa agg cca gtt ctg ccc cca cca ctgatc atc ttc agc cac 3536 His Glu Arg Pro Val Leu Pro Pro Pro Leu Ile IlePhe Ser His 1145 1150 1155 atg acc atg ata ttc cag cac ctg tgc tgc cgatgg agg aaa cac 3581 Met Thr Met Ile Phe Gln His Leu Cys Cys Arg Trp ArgLys His 1160 1165 1170 gag agc gac ccg gat gaa agg gac tac ggc ctg aaactc ttc ata 3626 Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu PheIle 1175 1180 1185 acc gat gat gag ctc aag aaa gta cat gac ttt gaa gagcaa tgc 3671 Thr Asp Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys1190 1195 1200 ata gaa gaa tac ttc aga gaa aag gat gat cgg ttc aac tcatct 3716 Ile Glu Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser1205 1210 1215 aat gat gag agg ata cgg gtg act tca gaa agg gtg gag aacatg 3761 Asn Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met1220 1225 1230 tct atg cgg ctg gag gaa gtc aac gag aga gag cac tcc atgaag 3806 Ser Met Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys1235 1240 1245 gct tca ctc cag acc gtg gac atc cgg ctg gcg cag ctg gaagac 3851 Ala Ser Leu Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp1250 1255 1260 ctt atc ggg cgc atg gcc acg gcc ctg gag cgc ctg aca ggtctg 3896 Leu Ile Gly Arg Met Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu1265 1270 1275 gag cgg gcc gag tcc aac aaa atc cgc tcg agg acc tcg tcagac 3941 Glu Arg Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp1280 1285 1290 tgc acg gac gcc gcc tac att gtc cgt cag agc agc ttc aacagc 3986 Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser1295 1300 1305 cag gaa ggg aac acc ttc aag ctc caa gag agt ata gac cctgca 4031 Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala1310 1315 1320 ggt gag gag acc atg tcc cca act tct cca acc tta atg ccccgt 4076 Gly Glu Glu Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg1325 1330 1335 atg cga agc cat tct ttc tat tca gtc aat atg aaa gac aaaggt 4121 Met Arg Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly1340 1345 1350 ggt ata gaa aag ttg gaa agt att ttt aaa gaa agg tcc ctgagc 4166 Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser1355 1360 1365 cta cac cgg gct act agt tcc cac tct gta gca aaa gaa cccaaa 4211 Leu His Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys1370 1375 1380 gct cct gca gcc cct gcc aac acc ttg gcc att gtt cct gattcc 4256 Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser1385 1390 1395 aga aga cca tca tcg tgt ata gac atc tat gtc tct gct atggat 4301 Arg Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp1400 1405 1410 gag ctc cac tgt gat ata gac cct ctg gac aat tcc gtg aacatc 4346 Glu Leu His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile1415 1420 1425 ctt ggg cta ggc gag cca agc ttt tca act cca gta cct tccaca 4391 Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr1430 1435 1440 gcc cct tca agt agt gcc tat gca aca ctt gca ccc aca gacaga 4436 Ala Pro Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg1445 1450 1455 cct cca agc cgg agc att gat ttt gag gac atc acc tcc atggac 4481 Pro Pro Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp1460 1465 1470 act aga tct ttt tct tca gac tac acc cac ctc cca gaa tgccaa 4526 Thr Arg Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln1475 1480 1485 aac ccc tgg gac tca gag cct ccg atg tac cac acc att gagcgt 4571 Asn Pro Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg1490 1495 1500 tcc aaa agt agc cgc tac cta gcc acc aca ccc ttt ctt ctagaa 4616 Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu1505 1510 1515 gag gct ccc att gtg aaa tct cat agc ttt atg ttt tcc ccctca 4661 Glu Ala Pro Ile Val Lys Ser His Ser Phe Met Phe Ser Pro Ser1520 1525 1530 agg agc tat tat gcc aac ttt ggg gtg cct gta aaa aca gcagaa 4706 Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala Glu1535 1540 1545 tac aca agt att aca gac tgt att gac aca agg tgt gtc aatgcc 4751 Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala1550 1555 1560 cct caa gca att gcg gac aga gct gcc ttc cct gga ggt cttgga 4796 Pro Gln Ala Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly1565 1570 1575 gac aaa gtg gag gac tta act tgc tgc cat cca gag cga gaagca 4841 Asp Lys Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala1580 1585 1590 gaa ctg agt cac ccc agc tct gac agt gag gag aat gag gccaaa 4886 Glu Leu Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys1595 1600 1605 ggc cgc aga gcc acc att gca ata tcc tcc cag gag ggt gataac 4931 Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn1610 1615 1620 tca gag aga acc ctg tcc aac aac atc act gtt ccc aag atagag 4976 Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu1625 1630 1635 cgc gcc aac agc tac tcg gca gag gag cca agt gcg cca tatgca 5021 Arg Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala1640 1645 1650 cac acc agg aag agc ttc tcc atc agt gac aaa ctc gac aggcag 5066 His Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln1655 1660 1665 cgg aac aca gca agc ctg caa aat ccc ttc cag aga agc aagtcc 5111 Arg Asn Thr Ala Ser Leu Gln Asn Pro Phe Gln Arg Ser Lys Ser1670 1675 1680 tcc aag ccg gag ggc cga ggg gac agc ctg tcc atg agg agactg 5156 Ser Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu1685 1690 1695 tcc aga aca tcg gct ttc caa agc ttt gaa agc aag cac acctaa 5201 Ser Arg Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His Thr 17001705 2 1709 PRT Homo sapiens 2 Met Gly Lys Lys Trp Arg Asp Ala Ala GluMet Glu Arg Gly Cys Ser 1 5 10 15 Asp Arg Glu Asp Asn Ala Glu Ser ArgArg Arg Ser Arg Ser Ala Ser 20 25 30 Arg Gly Arg Phe Ala Glu Ser Trp LysArg Leu Ser Ser Lys Gln Gly 35 40 45 Ser Thr Lys Arg Ser Gly Leu Pro SerGln Gln Thr Pro Ala Gln Lys 50 55 60 Ser Trp Ile Glu Arg Ala Phe Tyr LysArg Glu Cys Val His Ile Ile 65 70 75 80 Pro Ser Thr Lys Asp Pro His ArgCys Cys Cys Gly Arg Leu Ile Gly 85 90 95 Gln His Val Gly Leu Thr Pro SerIle Ser Val Leu Gln Asn Glu Lys 100 105 110 Asn Glu Ser Arg Leu Ser ArgAsn Asp Ile Gln Ser Glu Lys Trp Ser 115 120 125 Ile Ser Lys His Thr GlnLeu Ser Pro Thr Asp Ala Phe Gly Thr Ile 130 135 140 Glu Phe Gln Gly GlyGly His Ser Asn Lys Ala Met Tyr Val Arg Val 145 150 155 160 Ser Phe AspThr Lys Pro Asp Leu Leu Leu His Leu Met Thr Lys Glu 165 170 175 Trp GlnLeu Glu Leu Pro Lys Leu Leu Ile Ser Val His Gly Gly Leu 180 185 190 GlnAsn Phe Glu Leu Gln Pro Lys Leu Lys Gln Val Phe Gly Lys Gly 195 200 205Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly 210 215220 Val Asn Thr Gly Val Ile Arg His Val Gly Asp Ala Leu Lys Asp His 225230 235 240 Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala ProTrp 245 250 255 Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp ValVal Arg 260 265 270 Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu ThrVal Leu Asn 275 280 285 Ser Met His Ser His Phe Ile Leu Ala Asp Asn GlyThr Thr Gly Lys 290 295 300 Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln LeuGlu Lys His Ile Ser 305 310 315 320 Leu Gln Lys Ile Asn Thr Arg Ile GlyGln Gly Val Pro Val Val Ala 325 330 335 Leu Ile Val Glu Gly Gly Pro AsnVal Ile Ser Ile Val Leu Glu Tyr 340 345 350 Leu Arg Asp Thr Pro Pro ValPro Val Val Val Cys Asp Gly Ser Gly 355 360 365 Arg Ala Ser Asp Ile LeuAla Phe Gly His Lys Tyr Ser Glu Glu Gly 370 375 380 Gly Leu Ile Asn GluSer Leu Arg Asp Gln Leu Leu Val Thr Ile Gln 385 390 395 400 Lys Thr PheThr Tyr Thr Arg Thr Gln Ala Gln His Leu Phe Ile Ile 405 410 415 Leu MetGlu Cys Met Lys Lys Lys Glu Leu Ile Thr Val Phe Arg Met 420 425 430 GlySer Glu Gly His Gln Asp Ile Asp Leu Ala Ile Leu Thr Ala Leu 435 440 445Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu 450 455460 Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly 465470 475 480 Gln Gln Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp AlaLeu 485 490 495 Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu AsnGly Val 500 505 510 Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu GluLeu Tyr Asn 515 520 525 Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His LeuVal Arg Asp Val 530 535 540 Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg IleSer Leu Ile Asp Ile 545 550 555 560 Gly Leu Val Ile Glu Tyr Leu Met GlyGly Ala Tyr Arg Cys Asn Tyr 565 570 575 Thr Arg Lys Arg Phe Arg Thr LeuTyr His Asn Leu Phe Gly Pro Lys 580 585 590 Arg Pro Lys Ala Leu Lys LeuLeu Gly Met Glu Asp Asp Ile Pro Leu 595 600 605 Arg Arg Gly Arg Lys ThrThr Lys Lys Arg Glu Glu Glu Val Asp Ile 610 615 620 Asp Leu Asp Asp ProGlu Ile Asn His Phe Pro Phe Pro Phe His Glu 625 630 635 640 Leu Met ValTrp Ala Val Leu Met Lys Arg Gln Lys Met Ala Leu Phe 645 650 655 Phe TrpGln His Gly Glu Glu Ala Met Ala Lys Ala Leu Val Ala Cys 660 665 670 LysLeu Cys Lys Ala Met Ala His Glu Ala Ser Glu Asn Asp Met Val 675 680 685Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly Gln 690 695700 Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu 705710 715 720 Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn AlaThr 725 730 735 Cys Leu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe IleAla His 740 745 750 Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met GlyArg Leu Arg 755 760 765 Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu GlyIle Leu Leu Pro 770 775 780 Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn LysAsp Asp Met Pro Tyr 785 790 795 800 Met Ser Gln Ala Gln Glu Ile His LeuGln Glu Lys Glu Ala Glu Glu 805 810 815 Pro Glu Lys Pro Thr Lys Glu LysGlu Glu Glu Asp Met Glu Leu Thr 820 825 830 Ala Met Leu Gly Arg Asn AsnGly Glu Ser Ser Arg Lys Lys Asp Glu 835 840 845 Glu Glu Val Gln Ser LysHis Arg Leu Ile Pro Leu Gly Arg Lys Ile 850 855 860 Tyr Glu Phe Tyr AsnAla Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu 865 870 875 880 Ala Tyr IleGly Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys 885 890 895 Met GluArg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile 900 905 910 PheThr Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu Pro 915 920 925Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn 930 935940 Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu 945950 955 960 Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile TyrCys 965 970 975 Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile PheGly Val 980 985 990 Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly LysMet Met Ile 995 1000 1005 Asp Met Met Tyr Phe Val Ile Ile Met Leu ValVal Leu Met Ser 1010 1015 1020 Phe Gly Val Ala Arg Gln Ala Ile Leu PhePro Asn Glu Glu Pro 1025 1030 1035 Ser Trp Lys Leu Ala Lys Asn Ile PheTyr Met Pro Tyr Trp Met 1040 1045 1050 Ile Tyr Gly Glu Val Phe Ala AspGln Ile Asp Pro Pro Cys Gly 1055 1060 1065 Gln Asn Glu Thr Arg Glu AspGly Lys Ile Ile Gln Leu Pro Pro 1070 1075 1080 Cys Lys Thr Gly Ala TrpIle Val Pro Ala Ile Met Ala Cys Tyr 1085 1090 1095 Leu Leu Val Ala AsnIle Leu Leu Val Asn Leu Leu Ile Ala Val 1100 1105 1110 Phe Asn Asn ThrPhe Phe Glu Val Lys Ser Ile Ser Asn Gln Val 1115 1120 1125 Trp Lys PheGln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg 1130 1135 1140 Pro ValLeu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met 1145 1150 1155 IlePhe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser Asp 1160 1165 1170Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp 1175 11801185 Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu 11901195 1200 Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu1205 1210 1215 Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser MetArg 1220 1225 1230 Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys AlaSer Leu 1235 1240 1245 Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu AspLeu Ile Gly 1250 1255 1260 Arg Met Ala Thr Ala Leu Glu Arg Leu Thr GlyLeu Glu Arg Ala 1265 1270 1275 Glu Ser Asn Lys Ile Arg Ser Arg Thr SerSer Asp Cys Thr Asp 1280 1285 1290 Ala Ala Tyr Ile Val Arg Gln Ser SerPhe Asn Ser Gln Glu Gly 1295 1300 1305 Asn Thr Phe Lys Leu Gln Glu SerIle Asp Pro Ala Gly Glu Glu 1310 1315 1320 Thr Met Ser Pro Thr Ser ProThr Leu Met Pro Arg Met Arg Ser 1325 1330 1335 His Ser Phe Tyr Ser ValAsn Met Lys Asp Lys Gly Gly Ile Glu 1340 1345 1350 Lys Leu Glu Ser IlePhe Lys Glu Arg Ser Leu Ser Leu His Arg 1355 1360 1365 Ala Thr Ser SerHis Ser Val Ala Lys Glu Pro Lys Ala Pro Ala 1370 1375 1380 Ala Pro AlaAsn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro 1385 1390 1395 Ser SerCys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His 1400 1405 1410 CysAsp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu 1415 1420 1425Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser 1430 14351440 Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser 14451450 1455 Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser1460 1465 1470 Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn ProTrp 1475 1480 1485 Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg SerLys Ser 1490 1495 1500 Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu GluGlu Ala Pro 1505 1510 1515 Ile Val Lys Ser His Ser Phe Met Phe Ser ProSer Arg Ser Tyr 1520 1525 1530 Tyr Ala Asn Phe Gly Val Pro Val Lys ThrAla Glu Tyr Thr Ser 1535 1540 1545 Ile Thr Asp Cys Ile Asp Thr Arg CysVal Asn Ala Pro Gln Ala 1550 1555 1560 Ile Ala Asp Arg Ala Ala Phe ProGly Gly Leu Gly Asp Lys Val 1565 1570 1575 Glu Asp Leu Thr Cys Cys HisPro Glu Arg Glu Ala Glu Leu Ser 1580 1585 1590 His Pro Ser Ser Asp SerGlu Glu Asn Glu Ala Lys Gly Arg Arg 1595 1600 1605 Ala Thr Ile Ala IleSer Ser Gln Glu Gly Asp Asn Ser Glu Arg 1610 1615 1620 Thr Leu Ser AsnAsn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn 1625 1630 1635 Ser Tyr SerAla Glu Glu Pro Ser Ala Pro Tyr Ala His Thr Arg 1640 1645 1650 Lys SerPhe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr 1655 1660 1665 AlaSer Leu Gln Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro 1670 1675 1680Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg Thr 1685 16901695 Ser Ala Phe Gln Ser Phe Glu Ser Lys His Thr 1700 1705 3 5237 DNAHomo sapiens CDS (72)..(5234) 3 cgcccgcggc gaggagccag cgagagcgctcggcgctggg ctgtttcccg gccgagggag 60 gcgaacttct c atg ggg aag aag tgg agggat gcg gcg gaa atg gag cgg 110 Met Gly Lys Lys Trp Arg Asp Ala Ala GluMet Glu Arg 1 5 10 ggc tgc tcc gac cgc gag gac aac gcg gag agc cgc agacgc agc cgg 158 Gly Cys Ser Asp Arg Glu Asp Asn Ala Glu Ser Arg Arg ArgSer Arg 15 20 25 agc gcc agc cgg ggc agg ttt gcc gag tcg tgg aaa agg ttaagt tcc 206 Ser Ala Ser Arg Gly Arg Phe Ala Glu Ser Trp Lys Arg Leu SerSer 30 35 40 45 aag cag ggg tcc acc aaa cgc tcg gga ctc ccg tcg cag cagacg ccg 254 Lys Gln Gly Ser Thr Lys Arg Ser Gly Leu Pro Ser Gln Gln ThrPro 50 55 60 gct cag aaa tcc tgg ata gaa aga gca ttt tat aaa aga gaa tgtgtc 302 Ala Gln Lys Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val65 70 75 cac atc ata ccc agc acc aaa gac ccc cat agg tgt tgc tgt ggg cgt350 His Ile Ile Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly Arg 8085 90 ctg ata ggc cag cat gtt ggc ctc acc ccc agt atc tcc gtg ctt cag398 Leu Ile Gly Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu Gln 95100 105 aat gag aaa aat gaa agt cgc ctc tcc cga aat gac atc cag tct gaa446 Asn Glu Lys Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu 110115 120 125 aag tgg tcc atc agc aaa cac act caa ctc agc cct acg gat gctttt 494 Lys Trp Ser Ile Ser Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe130 135 140 ggg acc att gag ttc caa gga ggt ggc cat tcc aac aaa gcc atgtat 542 Gly Thr Ile Glu Phe Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr145 150 155 gtg cga gta tct ttt gat aca aaa cct gat ctc ctc tta cac ctgatg 590 Val Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met160 165 170 acc aag gaa tgg cag ttg gag ctt ccc aag ctt ctc atc tct gtccat 638 Thr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His175 180 185 ggg ggc ctg cag aac ttt gaa ctc cag cca aaa ctc aag caa gtcttt 686 Gly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln Val Phe190 195 200 205 ggg aaa ggg ctc atc aaa gca gca atg aca act gga gcg tggata ttc 734 Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp IlePhe 210 215 220 act gga ggg gtt aac aca ggt gtt att cgt cat gtt ggc gatgcc ttg 782 Thr Gly Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp AlaLeu 225 230 235 aag gat cat gcc tct aag tct cga gga aag ata tgc acc ataggt att 830 Lys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile GlyIle 240 245 250 gcc ccc tgg gga att gtg gaa aac cag gag gac ctc att ggaaga gat 878 Ala Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly ArgAsp 255 260 265 gtt gtc cgg cca tac cag acc atg tcc aat ccc atg agc aagctc act 926 Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys LeuThr 270 275 280 285 gtt ctc aac agc atg cat tcc cac ttc att ctg gct gacaac ggg acc 974 Val Leu Asn Ser Met His Ser His Phe Ile Leu Ala Asp AsnGly Thr 290 295 300 act gga aaa tat gga gca gag gtg aaa ctt cga aga caactg gaa aag 1022 Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln LeuGlu Lys 305 310 315 cat att tca ctc cag aag ata aac aca aga atc ggt caaggt gtt cct 1070 His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln GlyVal Pro 320 325 330 gtg gtg gca ctc ata gtg gaa gga gga ccc aat gtg atctcg att gtt 1118 Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile SerIle Val 335 340 345 ttg gag tac ctt cga gac acc cct ccc gtg cca gtg gttgtc tgt gat 1166 Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val ValCys Asp 350 355 360 365 ggg agt gga cgg gca tcg gac atc ctg gcc ttt gggcat aaa tac tca 1214 Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly HisLys Tyr Ser 370 375 380 gaa gaa ggc gga ctg ata aat gaa tct ttg agg gaccag ctg ttg gtg 1262 Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu Arg Asp GlnLeu Leu Val 385 390 395 act ata cag aag act ttc aca tac act cga acc caagct cag cat ctg 1310 Thr Ile Gln Lys Thr Phe Thr Tyr Thr Arg Thr Gln AlaGln His Leu 400 405 410 ttc atc atc ctc atg gag tgc atg aag aag aag gaattg att acg gta 1358 Phe Ile Ile Leu Met Glu Cys Met Lys Lys Lys Glu LeuIle Thr Val 415 420 425 ttt cgg atg gga tca gaa gga cac cag gac att gatttg gct atc ctg 1406 Phe Arg Met Gly Ser Glu Gly His Gln Asp Ile Asp LeuAla Ile Leu 430 435 440 445 aca gct tta ctc aaa gga gcc aat gcc tcg gcccca gac caa ctg agc 1454 Thr Ala Leu Leu Lys Gly Ala Asn Ala Ser Ala ProAsp Gln Leu Ser 450 455 460 tta gct tta gcc tgg aac aga gtc gac atc gctcgc agc cag atc ttt 1502 Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala ArgSer Gln Ile Phe 465 470 475 att tac ggg caa cag tgg ccg gtg gga tct ctggag caa gcc atg ttg 1550 Ile Tyr Gly Gln Gln Trp Pro Val Gly Ser Leu GluGln Ala Met Leu 480 485 490 gat gcc tta gtt ctg gac aga gtg gat ttt gtgaaa tta ctc ata gag 1598 Asp Ala Leu Val Leu Asp Arg Val Asp Phe Val LysLeu Leu Ile Glu 495 500 505 aat gga gta agc atg cac cgt ttt ctc acc atctcc aga cta gag gaa 1646 Asn Gly Val Ser Met His Arg Phe Leu Thr Ile SerArg Leu Glu Glu 510 515 520 525 ttg tac aat acg aga cat ggg ccc tca aataca ttg tac cac ttg gtc 1694 Leu Tyr Asn Thr Arg His Gly Pro Ser Asn ThrLeu Tyr His Leu Val 530 535 540 agg gat gtc aaa aag cga gag tat cca ggtttc ggt tgg atc tat ttt 1742 Arg Asp Val Lys Lys Arg Glu Tyr Pro Gly PheGly Trp Ile Tyr Phe 545 550 555 aag ggg aac ctg ccc cca gac tac aga atcagc ctg att gac atc ggc 1790 Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile SerLeu Ile Asp Ile Gly 560 565 570 ctg gtg atc gag tac ctg atg ggc ggg gcttat cgc tgc aac tac acg 1838 Leu Val Ile Glu Tyr Leu Met Gly Gly Ala TyrArg Cys Asn Tyr Thr 575 580 585 cgc aag cgc ttc cgg acc ctc tac cac aacctc ttc ggc ccc aag agg 1886 Arg Lys Arg Phe Arg Thr Leu Tyr His Asn LeuPhe Gly Pro Lys Arg 590 595 600 605 ccc aaa gcc ttg aaa ctg ctg gga atggag gat gat att ccc ttg agg 1934 Pro Lys Ala Leu Lys Leu Leu Gly Met GluAsp Asp Ile Pro Leu Arg 610 615 620 cga gga aga aag aca acc aag aaa cgtgaa gaa gag gtg gac att gac 1982 Arg Gly Arg Lys Thr Thr Lys Lys Arg GluGlu Glu Val Asp Ile Asp 625 630 635 ttg gat gat cct gag atc aac cac ttcccc ttc cct ttc cat gag ctc 2030 Leu Asp Asp Pro Glu Ile Asn His Phe ProPhe Pro Phe His Glu Leu 640 645 650 atg gtg tgg gct gtt ctc atg aag cggcag aag atg gcc ctg ttc ttc 2078 Met Val Trp Ala Val Leu Met Lys Arg GlnLys Met Ala Leu Phe Phe 655 660 665 tgg cag cac ggt gag gag gcc atg gccaag gcc ctg gtg gcc tgc aag 2126 Trp Gln His Gly Glu Glu Ala Met Ala LysAla Leu Val Ala Cys Lys 670 675 680 685 ctc tgc aaa gcc atg gct cat gaggcc tct gag aac gac atg gtt gac 2174 Leu Cys Lys Ala Met Ala His Glu AlaSer Glu Asn Asp Met Val Asp 690 695 700 gac att tcc cag gag ctg aat cacaat tcc aga gac ttt ggc cag ctg 2222 Asp Ile Ser Gln Glu Leu Asn His AsnSer Arg Asp Phe Gly Gln Leu 705 710 715 gct gtg gag ctc ctg gac cag tcctac aag cag gac gaa cag ctg gcc 2270 Ala Val Glu Leu Leu Asp Gln Ser TyrLys Gln Asp Glu Gln Leu Ala 720 725 730 atg aaa ctg ctg acg tat gag ctgaag aac tgg agc aac gcc acg tgc 2318 Met Lys Leu Leu Thr Tyr Glu Leu LysAsn Trp Ser Asn Ala Thr Cys 735 740 745 ctg cag ctt gcc gtg gct gcc aaacac cgc gac ttc atc gcg cac acg 2366 Leu Gln Leu Ala Val Ala Ala Lys HisArg Asp Phe Ile Ala His Thr 750 755 760 765 tgc agc cag atg ctg ctc accgac atg tgg atg ggc cgg ctc cgc atg 2414 Cys Ser Gln Met Leu Leu Thr AspMet Trp Met Gly Arg Leu Arg Met 770 775 780 cgc aag aac tca ggc ctc aaggta att ctg gga att cta ctt cct cct 2462 Arg Lys Asn Ser Gly Leu Lys ValIle Leu Gly Ile Leu Leu Pro Pro 785 790 795 tca att ctc agc ttg gag ttcaag aac aaa gac gac atg ccc tat atg 2510 Ser Ile Leu Ser Leu Glu Phe LysAsn Lys Asp Asp Met Pro Tyr Met 800 805 810 tct cag gcc cag gaa atc cacctc caa gag aag gag gca gaa gaa cca 2558 Ser Gln Ala Gln Glu Ile His LeuGln Glu Lys Glu Ala Glu Glu Pro 815 820 825 gag aag ccc aca aag gaa aaagag gaa gag gac atg gag ctc aca gca 2606 Glu Lys Pro Thr Lys Glu Lys GluGlu Glu Asp Met Glu Leu Thr Ala 830 835 840 845 atg ttg gga cga aac aacggg gag tcc tcc agg aag aag gat gaa gag 2654 Met Leu Gly Arg Asn Asn GlyGlu Ser Ser Arg Lys Lys Asp Glu Glu 850 855 860 gaa gtt cag agc aag caccgg tta atc ccc ctc ggc aga aaa atc tat 2702 Glu Val Gln Ser Lys His ArgLeu Ile Pro Leu Gly Arg Lys Ile Tyr 865 870 875 gaa ttc tac aat gca cccatc gtg aag ttc tgg ttc tac aca ctg gcg 2750 Glu Phe Tyr Asn Ala Pro IleVal Lys Phe Trp Phe Tyr Thr Leu Ala 880 885 890 tat atc gga tac ctg atgctc ttc aac tat atc gtg tta gtg aag atg 2798 Tyr Ile Gly Tyr Leu Met LeuPhe Asn Tyr Ile Val Leu Val Lys Met 895 900 905 gaa cgc tgg ccg tcc acccag gaa tgg atc gta atc tcc tat att ttc 2846 Glu Arg Trp Pro Ser Thr GlnGlu Trp Ile Val Ile Ser Tyr Ile Phe 910 915 920 925 acc ctg gga ata gaaaag atg aga gag att ctg atg tca gag cca ggg 2894 Thr Leu Gly Ile Glu LysMet Arg Glu Ile Leu Met Ser Glu Pro Gly 930 935 940 aag ttg cta cag aaagtg aag gta tgg ctg cag gag tac tgg aat gtc 2942 Lys Leu Leu Gln Lys ValLys Val Trp Leu Gln Glu Tyr Trp Asn Val 945 950 955 acg gac ctc atc gccatc ctt ctg ttt tct gtc gga atg atc ctt cgt 2990 Thr Asp Leu Ile Ala IleLeu Leu Phe Ser Val Gly Met Ile Leu Arg 960 965 970 ctc caa gac cag cccttc agg agt gac ggg agg gtc atc tac tgc gtg 3038 Leu Gln Asp Gln Pro PheArg Ser Asp Gly Arg Val Ile Tyr Cys Val 975 980 985 aac atc att tac tggtat atc cgt ctc cta gac atc ttc ggc gtg aac 3086 Asn Ile Ile Tyr Trp TyrIle Arg Leu Leu Asp Ile Phe Gly Val Asn 990 995 1000 1005 aag tat ttgggc ccg tat gta atg atg att gga aaa atg atg ata 3131 Lys Tyr Leu Gly ProTyr Val Met Met Ile Gly Lys Met Met Ile 1010 1015 1020 gac atg atg tacttt gtc atc att atg ctg gtg gtt ctg atg agc 3176 Asp Met Met Tyr Phe ValIle Ile Met Leu Val Val Leu Met Ser 1025 1030 1035 ttt ggg gtc gcc aggcaa gcc atc ctt ttt ccc aat gag gag cca 3221 Phe Gly Val Ala Arg Gln AlaIle Leu Phe Pro Asn Glu Glu Pro 1040 1045 1050 tca tgg aaa ctg gcc aagaac atc ttc tac atg ccc tat tgg atg 3266 Ser Trp Lys Leu Ala Lys Asn IlePhe Tyr Met Pro Tyr Trp Met 1055 1060 1065 att tat ggg gaa gtg ttt gcggac cag ata gac cct ccc tgt gga 3311 Ile Tyr Gly Glu Val Phe Ala Asp GlnIle Asp Pro Pro Cys Gly 1070 1075 1080 cag aat gag acc cga gag gat ggtaaa ata atc cag ctg cct ccc 3356 Gln Asn Glu Thr Arg Glu Asp Gly Lys IleIle Gln Leu Pro Pro 1085 1090 1095 tgc aag aca gga gct tgg atc gtg ccggcc atc atg gcc tgc tac 3401 Cys Lys Thr Gly Ala Trp Ile Val Pro Ala IleMet Ala Cys Tyr 1100 1105 1110 ctc tta gtg gca aac atc ttg ctg gtc aacctc ctc att gct gtc 3446 Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu LeuIle Ala Val 1115 1120 1125 ttt aac aat aca ttt ttt gaa gta aaa tcg atatcc aac caa gtc 3491 Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser AsnGln Val 1130 1135 1140 tgg aag ttt cag agg tat cag ctc atc atg act ttccat gaa agg 3536 Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His GluArg 1145 1150 1155 cca gtt ctg ccc cca cca ctg atc atc ttc agc cac atgacc atg 3581 Pro Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met1160 1165 1170 ata ttc cag cac ctg tgc tgc cga tgg agg aaa cac gag agcgac 3626 Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser Asp1175 1180 1185 ccg gat gaa agg gac tac ggc ctg aaa ctc ttc ata acc gatgat 3671 Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp1190 1195 1200 gag ctc aag aaa gta cat gac ttt gaa gag caa tgc ata gaagaa 3716 Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu1205 1210 1215 tac ttc aga gaa aag gat gat cgg ttc aac tca tct aat gatgag 3761 Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu1220 1225 1230 agg ata cgg gtg act tca gaa agg gtg gag aac atg tct atgcgg 3806 Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg1235 1240 1245 ctg gag gaa gtc aac gag aga gag cac tcc atg aag gct tcactc 3851 Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu1250 1255 1260 cag acc gtg gac atc cgg ctg gcg cag ctg gaa gac ctt atcggg 3896 Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly1265 1270 1275 cgc atg gcc acg gcc ctg gag cgc ctg aca ggt ctg gag cgggcc 3941 Arg Met Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala1280 1285 1290 gag tcc aac aaa atc cgc tcg agg acc tcg tca gac tgc acggac 3986 Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp1295 1300 1305 gcc gcc tac att gtc cgt cag agc agc ttc aac agc cag gaaggg 4031 Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly1310 1315 1320 aac acc ttc aag ctc caa gag agt ata gac cct gca ggt gaggag 4076 Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu1325 1330 1335 acc atg tcc cca act tct cca acc tta atg ccc cgt atg cgaagc 4121 Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg Ser1340 1345 1350 cat tct ttc tat tca gtc aat atg aaa gac aaa ggt ggt atagaa 4166 His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile Glu1355 1360 1365 aag ttg gaa agt att ttt aaa gaa agg tcc ctg agc cta caccgg 4211 Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His Arg1370 1375 1380 gct act agt tcc cac tct gta gca aaa gaa ccc aaa gct cctgca 4256 Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro Ala1385 1390 1395 gcc cct gcc aac acc ttg gcc att gtt cct gat tcc aga agacca 4301 Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro1400 1405 1410 tca tcg tgt ata gac atc tat gtc tct gct atg gat gag ctccac 4346 Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His1415 1420 1425 tgt gat ata gac cct ctg gac aat tcc gtg aac atc ctt gggcta 4391 Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu1430 1435 1440 ggc gag cca agc ttt tca act cca gta cct tcc aca gcc ccttca 4436 Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser1445 1450 1455 agt agt gcc tat gca aca ctt gca ccc aca gac aga cct ccaagc 4481 Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser1460 1465 1470 cgg agc att gat ttt gag gac atc acc tcc atg gac act agatct 4526 Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser1475 1480 1485 ttt tct tca gac tac acc cac ctc cca gaa tgc caa aac ccctgg 4571 Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp1490 1495 1500 gac tca gag cct ccg atg tac cac acc att gag cgt tcc aaaagt 4616 Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser1505 1510 1515 agc cgc tac cta gcc acc aca ccc ttt ctt cta gaa gag gctccc 4661 Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro1520 1525 1530 att gtg aaa tct cat agc ttt atg ttt tcc ccc tca agg agctat 4706 Ile Val Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr1535 1540 1545 tat gcc aac ttt ggg gtg cct gta aaa aca gca gaa tac acaagt 4751 Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser1550 1555 1560 att aca gac tgt att gac aca agg tgt gtc aat gcc cct caagca 4796 Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala1565 1570 1575 att gcg gac aga gct gcc ttc cct gga ggt ctt gga gac aaagtg 4841 Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys Val1580 1585 1590 gag gac tta act tgc tgc cat cca gag cga gaa gca gaa ctgagt 4886 Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu Ser1595 1600 1605 cac ccc agc tct gac agt gag gag aat gag gcc aaa ggc cgcaga 4931 His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg1610 1615 1620 gcc acc att gca ata tcc tcc cag gag ggt gat aac tca gagaga 4976 Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg1625 1630 1635 acc ctg tcc aac aac atc act gtt ccc aag ata gag cgc gccaac 5021 Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn1640 1645 1650 agc tac tcg gca gag gag cca agt gcg cca tat gca cac accagg 5066 Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr Arg1655 1660 1665 aag agc ttc tcc atc agt gac aaa ctc gac agg cag cgg aacaca 5111 Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr1670 1675 1680 gca agc ctg caa aat ccc ttc cag aga agc aag tcc tcc aagccg 5156 Ala Ser Leu Gln Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro1685 1690 1695 gag ggc cga ggg gac agc ctg tcc atg agg aga ctg tcc agaaca 5201 Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg Thr1700 1705 1710 tcg gct ttc caa agc ttt gaa agc aag cac acc taa 5237 SerAla Phe Gln Ser Phe Glu Ser Lys His Thr 1715 1720 4 1721 PRT Homosapiens 4 Met Gly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg Gly CysSer 1 5 10 15 Asp Arg Glu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg SerAla Ser 20 25 30 Arg Gly Arg Phe Ala Glu Ser Trp Lys Arg Leu Ser Ser LysGln Gly 35 40 45 Ser Thr Lys Arg Ser Gly Leu Pro Ser Gln Gln Thr Pro AlaGln Lys 50 55 60 Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val HisIle Ile 65 70 75 80 Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly ArgLeu Ile Gly 85 90 95 Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu GlnAsn Glu Lys 100 105 110 Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln SerGlu Lys Trp Ser 115 120 125 Ile Ser Lys His Thr Gln Leu Ser Pro Thr AspAla Phe Gly Thr Ile 130 135 140 Glu Phe Gln Gly Gly Gly His Ser Asn LysAla Met Tyr Val Arg Val 145 150 155 160 Ser Phe Asp Thr Lys Pro Asp LeuLeu Leu His Leu Met Thr Lys Glu 165 170 175 Trp Gln Leu Glu Leu Pro LysLeu Leu Ile Ser Val His Gly Gly Leu 180 185 190 Gln Asn Phe Glu Leu GlnPro Lys Leu Lys Gln Val Phe Gly Lys Gly 195 200 205 Leu Ile Lys Ala AlaMet Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly 210 215 220 Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu Lys Asp His 225 230 235 240 Ala SerLys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro Trp 245 250 255 GlyIle Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val Arg 260 265 270Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu Asn 275 280285 Ser Met His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr Gly Lys 290295 300 Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys His Ile Ser305 310 315 320 Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro ValVal Ala 325 330 335 Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser Ile ValLeu Glu Tyr 340 345 350 Leu Arg Asp Thr Pro Pro Val Pro Val Val Val CysAsp Gly Ser Gly 355 360 365 Arg Ala Ser Asp Ile Leu Ala Phe Gly His LysTyr Ser Glu Glu Gly 370 375 380 Gly Leu Ile Asn Glu Ser Leu Arg Asp GlnLeu Leu Val Thr Ile Gln 385 390 395 400 Lys Thr Phe Thr Tyr Thr Arg ThrGln Ala Gln His Leu Phe Ile Ile 405 410 415 Leu Met Glu Cys Met Lys LysLys Glu Leu Ile Thr Val Phe Arg Met 420 425 430 Gly Ser Glu Gly His GlnAsp Ile Asp Leu Ala Ile Leu Thr Ala Leu 435 440 445 Leu Lys Gly Ala AsnAla Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu 450 455 460 Ala Trp Asn ArgVal Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly 465 470 475 480 Gln GlnTrp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu 485 490 495 ValLeu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val 500 505 510Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr Asn 515 520525 Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp Val 530535 540 Lys Lys Arg Glu Tyr Pro Gly Phe Gly Trp Ile Tyr Phe Lys Gly Asn545 550 555 560 Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile Asp Ile Gly LeuVal Ile 565 570 575 Glu Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr ThrArg Lys Arg 580 585 590 Phe Arg Thr Leu Tyr His Asn Leu Phe Gly Pro LysArg Pro Lys Ala 595 600 605 Leu Lys Leu Leu Gly Met Glu Asp Asp Ile ProLeu Arg Arg Gly Arg 610 615 620 Lys Thr Thr Lys Lys Arg Glu Glu Glu ValAsp Ile Asp Leu Asp Asp 625 630 635 640 Pro Glu Ile Asn His Phe Pro PhePro Phe His Glu Leu Met Val Trp 645 650 655 Ala Val Leu Met Lys Arg GlnLys Met Ala Leu Phe Phe Trp Gln His 660 665 670 Gly Glu Glu Ala Met AlaLys Ala Leu Val Ala Cys Lys Leu Cys Lys 675 680 685 Ala Met Ala His GluAla Ser Glu Asn Asp Met Val Asp Asp Ile Ser 690 695 700 Gln Glu Leu AsnHis Asn Ser Arg Asp Phe Gly Gln Leu Ala Val Glu 705 710 715 720 Leu LeuAsp Gln Ser Tyr Lys Gln Asp Glu Gln Leu Ala Met Lys Leu 725 730 735 LeuThr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys Leu Gln Leu 740 745 750Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr Cys Ser Gln 755 760765 Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn 770775 780 Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu785 790 795 800 Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr Met SerGln Ala 805 810 815 Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu ProGlu Lys Pro 820 825 830 Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu ThrAla Met Leu Gly 835 840 845 Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys AspGlu Glu Glu Val Gln 850 855 860 Ser Lys His Arg Leu Ile Pro Leu Gly ArgLys Ile Tyr Glu Phe Tyr 865 870 875 880 Asn Ala Pro Ile Val Lys Phe TrpPhe Tyr Thr Leu Ala Tyr Ile Gly 885 890 895 Tyr Leu Met Leu Phe Asn TyrIle Val Leu Val Lys Met Glu Arg Trp 900 905 910 Pro Ser Thr Gln Glu TrpIle Val Ile Ser Tyr Ile Phe Thr Leu Gly 915 920 925 Ile Glu Lys Met ArgGlu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu 930 935 940 Gln Lys Val LysVal Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp Leu 945 950 955 960 Ile AlaIle Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln Asp 965 970 975 GlnPro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile Ile 980 985 990Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys Tyr Leu 995 10001005 Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met Met 10101015 1020 Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser Phe Gly Val1025 1030 1035 Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser TrpLys 1040 1045 1050 Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met IleTyr Gly 1055 1060 1065 Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys GlyGln Asn Glu 1070 1075 1080 Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu ProPro Cys Lys Thr 1085 1090 1095 Gly Ala Trp Ile Val Pro Ala Ile Met AlaCys Tyr Leu Leu Val 1100 1105 1110 Ala Asn Ile Leu Leu Val Asn Leu LeuIle Ala Val Phe Asn Asn 1115 1120 1125 Thr Phe Phe Glu Val Lys Ser IleSer Asn Gln Val Trp Lys Phe 1130 1135 1140 Gln Arg Tyr Gln Leu Ile MetThr Phe His Glu Arg Pro Val Leu 1145 1150 1155 Pro Pro Pro Leu Ile IlePhe Ser His Met Thr Met Ile Phe Gln 1160 1165 1170 His Leu Cys Cys ArgTrp Arg Lys His Glu Ser Asp Pro Asp Glu 1175 1180 1185 Arg Asp Tyr GlyLeu Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys 1190 1195 1200 Lys Val HisAsp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg 1205 1210 1215 Glu LysAsp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg 1220 1225 1230 ValThr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu 1235 1240 1245Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr Val 1250 12551260 Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala 12651270 1275 Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn1280 1285 1290 Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala AlaTyr 1295 1300 1305 Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly AsnThr Phe 1310 1315 1320 Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu GluThr Met Ser 1325 1330 1335 Pro Thr Ser Pro Thr Leu Met Pro Arg Met ArgSer His Ser Phe 1340 1345 1350 Tyr Ser Val Asn Met Lys Asp Lys Gly GlyIle Glu Lys Leu Glu 1355 1360 1365 Ser Ile Phe Lys Glu Arg Ser Leu SerLeu His Arg Ala Thr Ser 1370 1375 1380 Ser His Ser Val Ala Lys Glu ProLys Ala Pro Ala Ala Pro Ala 1385 1390 1395 Asn Thr Leu Ala Ile Val ProAsp Ser Arg Arg Pro Ser Ser Cys 1400 1405 1410 Ile Asp Ile Tyr Val SerAla Met Asp Glu Leu His Cys Asp Ile 1415 1420 1425 Asp Pro Leu Asp AsnSer Val Asn Ile Leu Gly Leu Gly Glu Pro 1430 1435 1440 Ser Phe Ser ThrPro Val Pro Ser Thr Ala Pro Ser Ser Ser Ala 1445 1450 1455 Tyr Ala ThrLeu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile 1460 1465 1470 Asp PheGlu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser 1475 1480 1485 AspTyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu 1490 1495 1500Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr 1505 15101515 Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys 15201525 1530 Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn1535 1540 1545 Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile ThrAsp 1550 1555 1560 Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala IleAla Asp 1565 1570 1575 Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys ValGlu Asp Leu 1580 1585 1590 Thr Cys Cys His Pro Glu Arg Glu Ala Glu LeuSer His Pro Ser 1595 1600 1605 Ser Asp Ser Glu Glu Asn Glu Ala Lys GlyArg Arg Ala Thr Ile 1610 1615 1620 Ala Ile Ser Ser Gln Glu Gly Asp AsnSer Glu Arg Thr Leu Ser 1625 1630 1635 Asn Asn Ile Thr Val Pro Lys IleGlu Arg Ala Asn Ser Tyr Ser 1640 1645 1650 Ala Glu Glu Pro Ser Ala ProTyr Ala His Thr Arg Lys Ser Phe 1655 1660 1665 Ser Ile Ser Asp Lys LeuAsp Arg Gln Arg Asn Thr Ala Ser Leu 1670 1675 1680 Gln Asn Pro Phe GlnArg Ser Lys Ser Ser Lys Pro Glu Gly Arg 1685 1690 1695 Gly Asp Ser LeuSer Met Arg Arg Leu Ser Arg Thr Ser Ala Phe 1700 1705 1710 Gln Ser PheGlu Ser Lys His Thr 1715 1720 5 5237 DNA Homo sapiens CDS (72)..(5234) 5cgcccgcggc gaggagccag cgagagcgct cggcgctggg ctgtttcccg gccgagggag 60gcgaacttct c atg ggg aag aag tgg agg gat gcg gcg gaa atg gag cgg 110 MetGly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg 1 5 10 ggc tgc tcc gaccgc gag gac aac gcg gag agc cgc aga cgc agc cgg 158 Gly Cys Ser Asp ArgGlu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg 15 20 25 agc gcc agc cgg ggcagg ttt gcc gag tcg tgg aaa agg tta agt tcc 206 Ser Ala Ser Arg Gly ArgPhe Ala Glu Ser Trp Lys Arg Leu Ser Ser 30 35 40 45 aag cag ggg tcc accaaa cgc tcg gga ctc ccg tcg cag cag acg ccg 254 Lys Gln Gly Ser Thr LysArg Ser Gly Leu Pro Ser Gln Gln Thr Pro 50 55 60 gct cag aaa tcc tgg atagaa aga gca ttt tat aaa aga gaa tgt gtc 302 Ala Gln Lys Ser Trp Ile GluArg Ala Phe Tyr Lys Arg Glu Cys Val 65 70 75 cac atc ata ccc agc acc aaagac ccc cat agg tgt tgc tgt ggg cgt 350 His Ile Ile Pro Ser Thr Lys AspPro His Arg Cys Cys Cys Gly Arg 80 85 90 ctg ata ggc cag cat gtt ggc ctcacc ccc agt atc tcc gtg ctt cag 398 Leu Ile Gly Gln His Val Gly Leu ThrPro Ser Ile Ser Val Leu Gln 95 100 105 aat gag aaa aat gaa agt cgc ctctcc cga aat gac atc cag tct gaa 446 Asn Glu Lys Asn Glu Ser Arg Leu SerArg Asn Asp Ile Gln Ser Glu 110 115 120 125 aag tgg tcc atc agc aaa cacact caa ctc agc cct acg gat gct ttt 494 Lys Trp Ser Ile Ser Lys His ThrGln Leu Ser Pro Thr Asp Ala Phe 130 135 140 ggg acc att gag ttc caa ggaggt ggc cat tcc aac aaa gcc atg tat 542 Gly Thr Ile Glu Phe Gln Gly GlyGly His Ser Asn Lys Ala Met Tyr 145 150 155 gtg cga gta tct ttt gat acaaaa cct gat ctc ctc tta cac ctg atg 590 Val Arg Val Ser Phe Asp Thr LysPro Asp Leu Leu Leu His Leu Met 160 165 170 acc aag gaa tgg cag ttg gagctt ccc aag ctt ctc atc tct gtc cat 638 Thr Lys Glu Trp Gln Leu Glu LeuPro Lys Leu Leu Ile Ser Val His 175 180 185 ggg ggc ctg cag aac ttt gaactc cag cca aaa ctc aag caa gtc ttt 686 Gly Gly Leu Gln Asn Phe Glu LeuGln Pro Lys Leu Lys Gln Val Phe 190 195 200 205 ggg aaa ggg ctc atc aaagca gca atg aca act gga gcg tgg ata ttc 734 Gly Lys Gly Leu Ile Lys AlaAla Met Thr Thr Gly Ala Trp Ile Phe 210 215 220 act gga ggg gtt aac acaggt gtt att cgt cat gtt ggc gat gcc ttg 782 Thr Gly Gly Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu 225 230 235 aag gat cat gcc tct aagtct cga gga aag ata tgc acc ata ggt att 830 Lys Asp His Ala Ser Lys SerArg Gly Lys Ile Cys Thr Ile Gly Ile 240 245 250 gcc ccc tgg gga att gtggaa aac cag gag gac ctc att gga aga gat 878 Ala Pro Trp Gly Ile Val GluAsn Gln Glu Asp Leu Ile Gly Arg Asp 255 260 265 gtt gtc cgg cca tac cagacc atg tcc aat ccc atg agc aag ctc act 926 Val Val Arg Pro Tyr Gln ThrMet Ser Asn Pro Met Ser Lys Leu Thr 270 275 280 285 gtt ctc aac agc atgcat tcc cac ttc att ctg gct gac aac ggg acc 974 Val Leu Asn Ser Met HisSer His Phe Ile Leu Ala Asp Asn Gly Thr 290 295 300 act gga aaa tat ggagca gag gtg aaa ctt cga aga caa ctg gaa aag 1022 Thr Gly Lys Tyr Gly AlaGlu Val Lys Leu Arg Arg Gln Leu Glu Lys 305 310 315 cat att tca ctc cagaag ata aac aca aga atc ggt caa ggt gtt cct 1070 His Ile Ser Leu Gln LysIle Asn Thr Arg Ile Gly Gln Gly Val Pro 320 325 330 gtg gtg gca ctc atagtg gaa gga gga ccc aat gtg atc tcg att gtt 1118 Val Val Ala Leu Ile ValGlu Gly Gly Pro Asn Val Ile Ser Ile Val 335 340 345 ttg gag tac ctt cgagac acc cct ccc gtg cca gtg gtt gtc tgt gat 1166 Leu Glu Tyr Leu Arg AspThr Pro Pro Val Pro Val Val Val Cys Asp 350 355 360 365 ggg agt gga cgggca tcg gac atc ctg gcc ttt ggg cat aaa tac tca 1214 Gly Ser Gly Arg AlaSer Asp Ile Leu Ala Phe Gly His Lys Tyr Ser 370 375 380 gaa gaa ggc ggactg ata aat gaa tct ttg agg gac cag ctg ttg gtg 1262 Glu Glu Gly Gly LeuIle Asn Glu Ser Leu Arg Asp Gln Leu Leu Val 385 390 395 act ata cag aagact ttc aca tac act cga acc caa gct cag cat ctg 1310 Thr Ile Gln Lys ThrPhe Thr Tyr Thr Arg Thr Gln Ala Gln His Leu 400 405 410 ttc atc atc ctcatg gag tgc atg aag aag aag gaa ttg att acg gta 1358 Phe Ile Ile Leu MetGlu Cys Met Lys Lys Lys Glu Leu Ile Thr Val 415 420 425 ttt cgg atg ggatca gaa gga cac cag gac att gat ttg gct atc ctg 1406 Phe Arg Met Gly SerGlu Gly His Gln Asp Ile Asp Leu Ala Ile Leu 430 435 440 445 aca gct ttactc aaa gga gcc aat gcc tcg gcc cca gac caa ctg agc 1454 Thr Ala Leu LeuLys Gly Ala Asn Ala Ser Ala Pro Asp Gln Leu Ser 450 455 460 tta gct ttagcc tgg aac aga gtc gac atc gct cgc agc cag atc ttt 1502 Leu Ala Leu AlaTrp Asn Arg Val Asp Ile Ala Arg Ser Gln Ile Phe 465 470 475 att tac gggcaa cag tgg ccg gtg gga tct ctg gag caa gcc atg ttg 1550 Ile Tyr Gly GlnGln Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu 480 485 490 gat gcc ttagtt ctg gac aga gtg gat ttt gtg aaa tta ctc ata gag 1598 Asp Ala Leu ValLeu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu 495 500 505 aat gga gtaagc atg cac cgt ttt ctc acc atc tcc aga cta gag gaa 1646 Asn Gly Val SerMet His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu 510 515 520 525 ttg tacaat acg aga cat ggg ccc tca aat aca ttg tac cac ttg gtc 1694 Leu Tyr AsnThr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val 530 535 540 agg gatgtc aaa aag ggg aac ctg ccc cca gac tac aga atc agc ctg 1742 Arg Asp ValLys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu 545 550 555 att gacatc ggc ctg gtg atc gag tac ctg atg ggc ggg gct tat cgc 1790 Ile Asp IleGly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg 560 565 570 tgc aactac acg cgc aag cgc ttc cgg acc ctc tac cac aac ctc ttc 1838 Cys Asn TyrThr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn Leu Phe 575 580 585 ggc cccaag agg ccc aaa gcc ttg aaa ctg ctg gga atg gag gat gat 1886 Gly Pro LysArg Pro Lys Ala Leu Lys Leu Leu Gly Met Glu Asp Asp 590 595 600 605 attccc ttg agg cga gga aga aag aca acc aag aaa cgt gaa gaa gag 1934 Ile ProLeu Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg Glu Glu Glu 610 615 620 gtggac att gac ttg gat gat cct gag atc aac cac ttc ccc ttc cct 1982 Val AspIle Asp Leu Asp Asp Pro Glu Ile Asn His Phe Pro Phe Pro 625 630 635 ttccat gag ctc atg gtg tgg gct gtt ctc atg aag cgg cag aag atg 2030 Phe HisGlu Leu Met Val Trp Ala Val Leu Met Lys Arg Gln Lys Met 640 645 650 gccctg ttc ttc tgg cag cac ggt gag gag gcc atg gcc aag gcc ctg 2078 Ala LeuPhe Phe Trp Gln His Gly Glu Glu Ala Met Ala Lys Ala Leu 655 660 665 gtggcc tgc aag ctc tgc aaa gcc atg gct cat gag gcc tct gag aac 2126 Val AlaCys Lys Leu Cys Lys Ala Met Ala His Glu Ala Ser Glu Asn 670 675 680 685gac atg gtt gac gac att tcc cag gag ctg aat cac aat tcc aga gac 2174 AspMet Val Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser Arg Asp 690 695 700ttt ggc cag ctg gct gtg gag ctc ctg gac cag tcc tac aag cag gac 2222 PheGly Gln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys Gln Asp 705 710 715gaa cag ctg gcc atg aaa ctg ctg acg tat gag ctg aag aac tgg agc 2270 GluGln Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser 720 725 730aac gcc acg tgc ctg cag ctt gcc gtg gct gcc aaa cac cgc gac ttc 2318 AsnAla Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe 735 740 745atc gcg cac acg tgc agc cag atg ctg ctc acc gac atg tgg atg ggc 2366 IleAla His Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly 750 755 760765 cgg ctc cgc atg cgc aag aac tca ggc ctc aag gta att ctg gga att 2414Arg Leu Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile 770 775780 cta ctt cct cct tca att ctc agc ttg gag ttc aag aac aaa gac gac 2462Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp 785 790795 atg ccc tat atg tct cag gcc cag gaa atc cac ctc caa gag aag gag 2510Met Pro Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu 800 805810 gca gaa gaa cca gag aag ccc aca aag gaa aaa gag gaa gag gac atg 2558Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp Met 815 820825 gag ctc aca gca atg ttg gga cga aac aac ggg gag tcc tcc agg aag 2606Glu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser Arg Lys 830 835840 845 aag gat gaa gag gaa gtt cag agc aag cac cgg tta atc ccc ctc ggc2654 Lys Asp Glu Glu Glu Val Gln Ser Lys His Arg Leu Ile Pro Leu Gly 850855 860 aga aaa atc tat gaa ttc tac aat gca ccc atc gtg aag ttc tgg ttc2702 Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe 865870 875 tac aca ctg gcg tat atc gga tac ctg atg ctc ttc aac tat atc gtg2750 Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val 880885 890 tta gtg aag atg gaa cgc tgg ccg tcc acc cag gaa tgg atc gta atc2798 Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile 895900 905 tcc tat att ttc acc ctg gga ata gaa aag atg aga gag att ctg atg2846 Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met 910915 920 925 tca gag cca ggg aag ttg cta cag aaa gtg aag gta tgg ctg caggag 2894 Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu930 935 940 tac tgg aat gtc acg gac ctc atc gcc atc ctt ctg ttt tct gtcgga 2942 Tyr Trp Asn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly945 950 955 atg atc ctt cgt ctc caa gac cag ccc ttc agg agt gac ggg agggtc 2990 Met Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val960 965 970 atc tac tgc gtg aac atc att tac tgg tat atc cgt ctc cta gacatc 3038 Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile975 980 985 ttc ggc gtg aac aag tat ttg ggc ccg tat gta atg atg att ggaaaa 3086 Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys990 995 1000 1005 atg atg ata gac atg atg tac ttt gtc atc att atg ctggtg gtt 3131 Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val Val1010 1015 1020 ctg atg agc ttt ggg gtc gcc agg caa gcc atc ctt ttt cccaat 3176 Leu Met Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn1025 1030 1035 gag gag cca tca tgg aaa ctg gcc aag aac atc ttc tac atgccc 3221 Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro1040 1045 1050 tat tgg atg att tat ggg gaa gtg ttt gcg gac cag ata gaccgt 3266 Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Arg1055 1060 1065 aag caa gtt tat gat tct cat aca cca aag tca gct ccc tgtgga 3311 Lys Gln Val Tyr Asp Ser His Thr Pro Lys Ser Ala Pro Cys Gly1070 1075 1080 cag aat gag acc cga gag gat ggt aaa ata atc cag ctg cctccc 3356 Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro1085 1090 1095 tgc aag aca gga gct tgg atc gtg ccg gcc atc atg gcc tgctac 3401 Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys Tyr1100 1105 1110 ctc tta gtg gca aac atc ttg ctg gtc aac ctc ctc att gctgtc 3446 Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Val1115 1120 1125 ttt aac aat aca ttt ttt gaa gta aaa tcg ata tcc aac caagtc 3491 Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val1130 1135 1140 tgg aag ttt cag agg tat cag ctc atc atg act ttc cat gaaagg 3536 Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg1145 1150 1155 cca gtt ctg ccc cca cca ctg atc atc ttc agc cac atg accatg 3581 Pro Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met1160 1165 1170 ata ttc cag cac ctg tgc tgc cga tgg agg aaa cac gag agcgac 3626 Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser Asp1175 1180 1185 ccg gat gaa agg gac tac ggc ctg aaa ctc ttc ata acc gatgat 3671 Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp1190 1195 1200 gag ctc aag aaa gta cat gac ttt gaa gag caa tgc ata gaagaa 3716 Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu1205 1210 1215 tac ttc aga gaa aag gat gat cgg ttc aac tca tct aat gatgag 3761 Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu1220 1225 1230 agg ata cgg gtg act tca gaa agg gtg gag aac atg tct atgcgg 3806 Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg1235 1240 1245 ctg gag gaa gtc aac gag aga gag cac tcc atg aag gct tcactc 3851 Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu1250 1255 1260 cag acc gtg gac atc cgg ctg gcg cag ctg gaa gac ctt atcggg 3896 Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly1265 1270 1275 cgc atg gcc acg gcc ctg gag cgc ctg aca ggt ctg gag cgggcc 3941 Arg Met Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala1280 1285 1290 gag tcc aac aaa atc cgc tcg agg acc tcg tca gac tgc acggac 3986 Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp1295 1300 1305 gcc gcc tac att gtc cgt cag agc agc ttc aac agc cag gaaggg 4031 Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly1310 1315 1320 aac acc ttc aag ctc caa gag agt ata gac cct gca ggt gaggag 4076 Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu1325 1330 1335 acc atg tcc cca act tct cca acc tta atg ccc cgt atg cgaagc 4121 Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg Ser1340 1345 1350 cat tct ttc tat tca gtc aat atg aaa gac aaa ggt ggt atagaa 4166 His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile Glu1355 1360 1365 aag ttg gaa agt att ttt aaa gaa agg tcc ctg agc cta caccgg 4211 Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His Arg1370 1375 1380 gct act agt tcc cac tct gta gca aaa gaa ccc aaa gct cctgca 4256 Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro Ala1385 1390 1395 gcc cct gcc aac acc ttg gcc att gtt cct gat tcc aga agacca 4301 Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro1400 1405 1410 tca tcg tgt ata gac atc tat gtc tct gct atg gat gag ctccac 4346 Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His1415 1420 1425 tgt gat ata gac cct ctg gac aat tcc gtg aac atc ctt gggcta 4391 Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu1430 1435 1440 ggc gag cca agc ttt tca act cca gta cct tcc aca gcc ccttca 4436 Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser1445 1450 1455 agt agt gcc tat gca aca ctt gca ccc aca gac aga cct ccaagc 4481 Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser1460 1465 1470 cgg agc att gat ttt gag gac atc acc tcc atg gac act agatct 4526 Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser1475 1480 1485 ttt tct tca gac tac acc cac ctc cca gaa tgc caa aac ccctgg 4571 Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp1490 1495 1500 gac tca gag cct ccg atg tac cac acc att gag cgt tcc aaaagt 4616 Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser1505 1510 1515 agc cgc tac cta gcc acc aca ccc ttt ctt cta gaa gag gctccc 4661 Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro1520 1525 1530 att gtg aaa tct cat agc ttt atg ttt tcc ccc tca agg agctat 4706 Ile Val Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr1535 1540 1545 tat gcc aac ttt ggg gtg cct gta aaa aca gca gaa tac acaagt 4751 Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser1550 1555 1560 att aca gac tgt att gac aca agg tgt gtc aat gcc cct caagca 4796 Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala1565 1570 1575 att gcg gac aga gct gcc ttc cct gga ggt ctt gga gac aaagtg 4841 Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys Val1580 1585 1590 gag gac tta act tgc tgc cat cca gag cga gaa gca gaa ctgagt 4886 Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu Ser1595 1600 1605 cac ccc agc tct gac agt gag gag aat gag gcc aaa ggc cgcaga 4931 His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg1610 1615 1620 gcc acc att gca ata tcc tcc cag gag ggt gat aac tca gagaga 4976 Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg1625 1630 1635 acc ctg tcc aac aac atc act gtt ccc aag ata gag cgc gccaac 5021 Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn1640 1645 1650 agc tac tcg gca gag gag cca agt gcg cca tat gca cac accagg 5066 Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr Arg1655 1660 1665 aag agc ttc tcc atc agt gac aaa ctc gac agg cag cgg aacaca 5111 Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr1670 1675 1680 gca agc ctg caa aat ccc ttc cag aga agc aag tcc tcc aagccg 5156 Ala Ser Leu Gln Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro1685 1690 1695 gag ggc cga ggg gac agc ctg tcc atg agg aga ctg tcc agaaca 5201 Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg Thr1700 1705 1710 tcg gct ttc caa agc ttt gaa agc aag cac acc taa 5237 SerAla Phe Gln Ser Phe Glu Ser Lys His Thr 1715 1720 6 1721 PRT Homosapiens 6 Met Gly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg Gly CysSer 1 5 10 15 Asp Arg Glu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg SerAla Ser 20 25 30 Arg Gly Arg Phe Ala Glu Ser Trp Lys Arg Leu Ser Ser LysGln Gly 35 40 45 Ser Thr Lys Arg Ser Gly Leu Pro Ser Gln Gln Thr Pro AlaGln Lys 50 55 60 Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val HisIle Ile 65 70 75 80 Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly ArgLeu Ile Gly 85 90 95 Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu GlnAsn Glu Lys 100 105 110 Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln SerGlu Lys Trp Ser 115 120 125 Ile Ser Lys His Thr Gln Leu Ser Pro Thr AspAla Phe Gly Thr Ile 130 135 140 Glu Phe Gln Gly Gly Gly His Ser Asn LysAla Met Tyr Val Arg Val 145 150 155 160 Ser Phe Asp Thr Lys Pro Asp LeuLeu Leu His Leu Met Thr Lys Glu 165 170 175 Trp Gln Leu Glu Leu Pro LysLeu Leu Ile Ser Val His Gly Gly Leu 180 185 190 Gln Asn Phe Glu Leu GlnPro Lys Leu Lys Gln Val Phe Gly Lys Gly 195 200 205 Leu Ile Lys Ala AlaMet Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly 210 215 220 Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu Lys Asp His 225 230 235 240 Ala SerLys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro Trp 245 250 255 GlyIle Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val Arg 260 265 270Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu Asn 275 280285 Ser Met His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr Gly Lys 290295 300 Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys His Ile Ser305 310 315 320 Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro ValVal Ala 325 330 335 Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser Ile ValLeu Glu Tyr 340 345 350 Leu Arg Asp Thr Pro Pro Val Pro Val Val Val CysAsp Gly Ser Gly 355 360 365 Arg Ala Ser Asp Ile Leu Ala Phe Gly His LysTyr Ser Glu Glu Gly 370 375 380 Gly Leu Ile Asn Glu Ser Leu Arg Asp GlnLeu Leu Val Thr Ile Gln 385 390 395 400 Lys Thr Phe Thr Tyr Thr Arg ThrGln Ala Gln His Leu Phe Ile Ile 405 410 415 Leu Met Glu Cys Met Lys LysLys Glu Leu Ile Thr Val Phe Arg Met 420 425 430 Gly Ser Glu Gly His GlnAsp Ile Asp Leu Ala Ile Leu Thr Ala Leu 435 440 445 Leu Lys Gly Ala AsnAla Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu 450 455 460 Ala Trp Asn ArgVal Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly 465 470 475 480 Gln GlnTrp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu 485 490 495 ValLeu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val 500 505 510Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr Asn 515 520525 Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp Val 530535 540 Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile Asp Ile545 550 555 560 Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg CysAsn Tyr 565 570 575 Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn Leu PheGly Pro Lys 580 585 590 Arg Pro Lys Ala Leu Lys Leu Leu Gly Met Glu AspAsp Ile Pro Leu 595 600 605 Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg GluGlu Glu Val Asp Ile 610 615 620 Asp Leu Asp Asp Pro Glu Ile Asn His PhePro Phe Pro Phe His Glu 625 630 635 640 Leu Met Val Trp Ala Val Leu MetLys Arg Gln Lys Met Ala Leu Phe 645 650 655 Phe Trp Gln His Gly Glu GluAla Met Ala Lys Ala Leu Val Ala Cys 660 665 670 Lys Leu Cys Lys Ala MetAla His Glu Ala Ser Glu Asn Asp Met Val 675 680 685 Asp Asp Ile Ser GlnGlu Leu Asn His Asn Ser Arg Asp Phe Gly Gln 690 695 700 Leu Ala Val GluLeu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu 705 710 715 720 Ala MetLys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr 725 730 735 CysLeu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His 740 745 750Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg 755 760765 Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro 770775 780 Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr785 790 795 800 Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu AlaGlu Glu 805 810 815 Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp MetGlu Leu Thr 820 825 830 Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser ArgLys Lys Asp Glu 835 840 845 Glu Glu Val Gln Ser Lys His Arg Leu Ile ProLeu Gly Arg Lys Ile 850 855 860 Tyr Glu Phe Tyr Asn Ala Pro Ile Val LysPhe Trp Phe Tyr Thr Leu 865 870 875 880 Ala Tyr Ile Gly Tyr Leu Met LeuPhe Asn Tyr Ile Val Leu Val Lys 885 890 895 Met Glu Arg Trp Pro Ser ThrGln Glu Trp Ile Val Ile Ser Tyr Ile 900 905 910 Phe Thr Leu Gly Ile GluLys Met Arg Glu Ile Leu Met Ser Glu Pro 915 920 925 Gly Lys Leu Leu GlnLys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn 930 935 940 Val Thr Asp LeuIle Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu 945 950 955 960 Arg LeuGln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys 965 970 975 ValAsn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val 980 985 990Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile 995 10001005 Asp Met Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser 10101015 1020 Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro1025 1030 1035 Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr TrpMet 1040 1045 1050 Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Arg LysGln Val 1055 1060 1065 Tyr Asp Ser His Thr Pro Lys Ser Ala Pro Cys GlyGln Asn Glu 1070 1075 1080 Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu ProPro Cys Lys Thr 1085 1090 1095 Gly Ala Trp Ile Val Pro Ala Ile Met AlaCys Tyr Leu Leu Val 1100 1105 1110 Ala Asn Ile Leu Leu Val Asn Leu LeuIle Ala Val Phe Asn Asn 1115 1120 1125 Thr Phe Phe Glu Val Lys Ser IleSer Asn Gln Val Trp Lys Phe 1130 1135 1140 Gln Arg Tyr Gln Leu Ile MetThr Phe His Glu Arg Pro Val Leu 1145 1150 1155 Pro Pro Pro Leu Ile IlePhe Ser His Met Thr Met Ile Phe Gln 1160 1165 1170 His Leu Cys Cys ArgTrp Arg Lys His Glu Ser Asp Pro Asp Glu 1175 1180 1185 Arg Asp Tyr GlyLeu Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys 1190 1195 1200 Lys Val HisAsp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg 1205 1210 1215 Glu LysAsp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg 1220 1225 1230 ValThr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu 1235 1240 1245Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr Val 1250 12551260 Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala 12651270 1275 Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn1280 1285 1290 Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala AlaTyr 1295 1300 1305 Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly AsnThr Phe 1310 1315 1320 Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu GluThr Met Ser 1325 1330 1335 Pro Thr Ser Pro Thr Leu Met Pro Arg Met ArgSer His Ser Phe 1340 1345 1350 Tyr Ser Val Asn Met Lys Asp Lys Gly GlyIle Glu Lys Leu Glu 1355 1360 1365 Ser Ile Phe Lys Glu Arg Ser Leu SerLeu His Arg Ala Thr Ser 1370 1375 1380 Ser His Ser Val Ala Lys Glu ProLys Ala Pro Ala Ala Pro Ala 1385 1390 1395 Asn Thr Leu Ala Ile Val ProAsp Ser Arg Arg Pro Ser Ser Cys 1400 1405 1410 Ile Asp Ile Tyr Val SerAla Met Asp Glu Leu His Cys Asp Ile 1415 1420 1425 Asp Pro Leu Asp AsnSer Val Asn Ile Leu Gly Leu Gly Glu Pro 1430 1435 1440 Ser Phe Ser ThrPro Val Pro Ser Thr Ala Pro Ser Ser Ser Ala 1445 1450 1455 Tyr Ala ThrLeu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile 1460 1465 1470 Asp PheGlu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser 1475 1480 1485 AspTyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu 1490 1495 1500Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr 1505 15101515 Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys 15201525 1530 Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn1535 1540 1545 Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile ThrAsp 1550 1555 1560 Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala IleAla Asp 1565 1570 1575 Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys ValGlu Asp Leu 1580 1585 1590 Thr Cys Cys His Pro Glu Arg Glu Ala Glu LeuSer His Pro Ser 1595 1600 1605 Ser Asp Ser Glu Glu Asn Glu Ala Lys GlyArg Arg Ala Thr Ile 1610 1615 1620 Ala Ile Ser Ser Gln Glu Gly Asp AsnSer Glu Arg Thr Leu Ser 1625 1630 1635 Asn Asn Ile Thr Val Pro Lys IleGlu Arg Ala Asn Ser Tyr Ser 1640 1645 1650 Ala Glu Glu Pro Ser Ala ProTyr Ala His Thr Arg Lys Ser Phe 1655 1660 1665 Ser Ile Ser Asp Lys LeuAsp Arg Gln Arg Asn Thr Ala Ser Leu 1670 1675 1680 Gln Asn Pro Phe GlnArg Ser Lys Ser Ser Lys Pro Glu Gly Arg 1685 1690 1695 Gly Asp Ser LeuSer Met Arg Arg Leu Ser Arg Thr Ser Ala Phe 1700 1705 1710 Gln Ser PheGlu Ser Lys His Thr 1715 1720 7 5171 DNA Homo sapiens CDS (72)..(5168) 7cgcccgcggc gaggagccag cgagagcgct cggcgctggg ctgtttcccg gccgagggag 60gcgaacttct c atg ggg aag aag tgg agg gat gcg gcg gaa atg gag cgg 110 MetGly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg 1 5 10 ggc tgc tcc gaccgc gag gac aac gcg gag agc cgc aga cgc agc cgg 158 Gly Cys Ser Asp ArgGlu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg 15 20 25 agc gcc agc cgg ggcagg ttt gcc gag tcg tgg aaa agg tta agt tcc 206 Ser Ala Ser Arg Gly ArgPhe Ala Glu Ser Trp Lys Arg Leu Ser Ser 30 35 40 45 aag cag ggg tcc accaaa cgc tcg gga ctc ccg tcg cag cag acg ccg 254 Lys Gln Gly Ser Thr LysArg Ser Gly Leu Pro Ser Gln Gln Thr Pro 50 55 60 gct cag aaa tcc tgg atagaa aga gca ttt tat aaa aga gaa tgt gtc 302 Ala Gln Lys Ser Trp Ile GluArg Ala Phe Tyr Lys Arg Glu Cys Val 65 70 75 cac atc ata ccc agc acc aaagac ccc cat agg tgt tgc tgt ggg cgt 350 His Ile Ile Pro Ser Thr Lys AspPro His Arg Cys Cys Cys Gly Arg 80 85 90 ctg ata ggc cag cat gtt ggc ctcacc ccc agt atc tcc gtg ctt cag 398 Leu Ile Gly Gln His Val Gly Leu ThrPro Ser Ile Ser Val Leu Gln 95 100 105 aat gag aaa aat gaa agt cgc ctctcc cga aat gac atc cag tct gaa 446 Asn Glu Lys Asn Glu Ser Arg Leu SerArg Asn Asp Ile Gln Ser Glu 110 115 120 125 aag tgg tcc atc agc aaa cacact caa ctc agc cct acg gat gct ttt 494 Lys Trp Ser Ile Ser Lys His ThrGln Leu Ser Pro Thr Asp Ala Phe 130 135 140 ggg acc att gag ttc caa ggaggt ggc cat tcc aac aaa gcc atg tat 542 Gly Thr Ile Glu Phe Gln Gly GlyGly His Ser Asn Lys Ala Met Tyr 145 150 155 gtg cga gta tct ttt gat acaaaa cct gat ctc ctc tta cac ctg atg 590 Val Arg Val Ser Phe Asp Thr LysPro Asp Leu Leu Leu His Leu Met 160 165 170 acc aag gaa tgg cag ttg gagctt ccc aag ctt ctc atc tct gtc cat 638 Thr Lys Glu Trp Gln Leu Glu LeuPro Lys Leu Leu Ile Ser Val His 175 180 185 ggg ggc ctg cag aac ttt gaactc cag cca aaa ctc aag caa gtc ttt 686 Gly Gly Leu Gln Asn Phe Glu LeuGln Pro Lys Leu Lys Gln Val Phe 190 195 200 205 ggg aaa ggg ctc atc aaagca gca atg aca act gga gcg tgg ata ttc 734 Gly Lys Gly Leu Ile Lys AlaAla Met Thr Thr Gly Ala Trp Ile Phe 210 215 220 act gga ggg gtt aac acaggt gtt att cgt cat gtt ggc gat gcc ttg 782 Thr Gly Gly Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu 225 230 235 aag gat cat gcc tct aagtct cga gga aag ata tgc acc ata ggt att 830 Lys Asp His Ala Ser Lys SerArg Gly Lys Ile Cys Thr Ile Gly Ile 240 245 250 gcc ccc tgg gga att gtggaa aac cag gag gac ctc att gga aga gat 878 Ala Pro Trp Gly Ile Val GluAsn Gln Glu Asp Leu Ile Gly Arg Asp 255 260 265 gtt gtc cgg cca tac cagacc atg tcc aat ccc atg agc aag ctc act 926 Val Val Arg Pro Tyr Gln ThrMet Ser Asn Pro Met Ser Lys Leu Thr 270 275 280 285 gtt ctc aac agc atgcat tcc cac ttc att ctg gct gac aac ggg acc 974 Val Leu Asn Ser Met HisSer His Phe Ile Leu Ala Asp Asn Gly Thr 290 295 300 act gga aaa tat ggagca gag gtg aaa ctt cga aga caa ctg gaa aag 1022 Thr Gly Lys Tyr Gly AlaGlu Val Lys Leu Arg Arg Gln Leu Glu Lys 305 310 315 cat att tca ctc cagaag ata aac aca aga atc ggt caa ggt gtt cct 1070 His Ile Ser Leu Gln LysIle Asn Thr Arg Ile Gly Gln Gly Val Pro 320 325 330 gtg gtg gca ctc atagtg gaa gga gga ccc aat gtg atc tcg att gtt 1118 Val Val Ala Leu Ile ValGlu Gly Gly Pro Asn Val Ile Ser Ile Val 335 340 345 ttg gag tac ctt cgagac acc cct ccc gtg cca gtg gtt gtc tgt gat 1166 Leu Glu Tyr Leu Arg AspThr Pro Pro Val Pro Val Val Val Cys Asp 350 355 360 365 ggg agt gga cgggca tcg gac atc ctg gcc ttt ggg cat aaa tac tca 1214 Gly Ser Gly Arg AlaSer Asp Ile Leu Ala Phe Gly His Lys Tyr Ser 370 375 380 gaa gaa ggc ggactg ata aat gaa tct ttg agg gac cag ctg ttg gtg 1262 Glu Glu Gly Gly LeuIle Asn Glu Ser Leu Arg Asp Gln Leu Leu Val 385 390 395 act ata cag aagact ttc aca tac act cga acc caa gct cag cat ctg 1310 Thr Ile Gln Lys ThrPhe Thr Tyr Thr Arg Thr Gln Ala Gln His Leu 400 405 410 ttc atc atc ctcatg gag tgc atg aag aag aag gaa ttg att acg gta 1358 Phe Ile Ile Leu MetGlu Cys Met Lys Lys Lys Glu Leu Ile Thr Val 415 420 425 ttt cgg atg ggatca gaa gga cac cag gac att gat ttg gct atc ctg 1406 Phe Arg Met Gly SerGlu Gly His Gln Asp Ile Asp Leu Ala Ile Leu 430 435 440 445 aca gct ttactc aaa gga gcc aat gcc tcg gcc cca gac caa ctg agc 1454 Thr Ala Leu LeuLys Gly Ala Asn Ala Ser Ala Pro Asp Gln Leu Ser 450 455 460 tta gct ttagcc tgg aac aga gtc gac atc gct cgc agc cag atc ttt 1502 Leu Ala Leu AlaTrp Asn Arg Val Asp Ile Ala Arg Ser Gln Ile Phe 465 470 475 att tac gggcaa cag tgg ccg gtg gga tct ctg gag caa gcc atg ttg 1550 Ile Tyr Gly GlnGln Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu 480 485 490 gat gcc ttagtt ctg gac aga gtg gat ttt gtg aaa tta ctc ata gag 1598 Asp Ala Leu ValLeu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu 495 500 505 aat gga gtaagc atg cac cgt ttt ctc acc atc tcc aga cta gag gaa 1646 Asn Gly Val SerMet His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu 510 515 520 525 ttg tacaat acg aga cat ggg ccc tca aat aca ttg tac cac ttg gtc 1694 Leu Tyr AsnThr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val 530 535 540 agg gatgtc aaa aag ggg aac ctg ccc cca gac tac aga atc agc ctg 1742 Arg Asp ValLys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu 545 550 555 att gacatc ggc ctg gtg atc gag tac ctg atg ggc ggg gct tat cgc 1790 Ile Asp IleGly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg 560 565 570 tgc aactac acg cgc aag cgc ttc cgg acc ctc tac cac aac ctc ttc 1838 Cys Asn TyrThr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn Leu Phe 575 580 585 ggc cccaag agg gat gat att ccc ttg agg cga gga aga aag aca acc 1886 Gly Pro LysArg Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys Thr Thr 590 595 600 605 aagaaa cgt gaa gaa gag gtg gac att gac ttg gat gat cct gag atc 1934 Lys LysArg Glu Glu Glu Val Asp Ile Asp Leu Asp Asp Pro Glu Ile 610 615 620 aaccac ttc ccc ttc cct ttc cat gag ctc atg gtg tgg gct gtt ctc 1982 Asn HisPhe Pro Phe Pro Phe His Glu Leu Met Val Trp Ala Val Leu 625 630 635 atgaag cgg cag aag atg gcc ctg ttc ttc tgg cag cac ggt gag gag 2030 Met LysArg Gln Lys Met Ala Leu Phe Phe Trp Gln His Gly Glu Glu 640 645 650 gccatg gcc aag gcc ctg gtg gcc tgc aag ctc tgc aaa gcc atg gct 2078 Ala MetAla Lys Ala Leu Val Ala Cys Lys Leu Cys Lys Ala Met Ala 655 660 665 catgag gcc tct gag aac gac atg gtt gac gac att tcc cag gag ctg 2126 His GluAla Ser Glu Asn Asp Met Val Asp Asp Ile Ser Gln Glu Leu 670 675 680 685aat cac aat tcc aga gac ttt ggc cag ctg gct gtg gag ctc ctg gac 2174 AsnHis Asn Ser Arg Asp Phe Gly Gln Leu Ala Val Glu Leu Leu Asp 690 695 700cag tcc tac aag cag gac gaa cag ctg gcc atg aaa ctg ctg acg tat 2222 GlnSer Tyr Lys Gln Asp Glu Gln Leu Ala Met Lys Leu Leu Thr Tyr 705 710 715gag ctg aag aac tgg agc aac gcc acg tgc ctg cag ctt gcc gtg gct 2270 GluLeu Lys Asn Trp Ser Asn Ala Thr Cys Leu Gln Leu Ala Val Ala 720 725 730gcc aaa cac cgc gac ttc atc gcg cac acg tgc agc cag atg ctg ctc 2318 AlaLys His Arg Asp Phe Ile Ala His Thr Cys Ser Gln Met Leu Leu 735 740 745acc gac atg tgg atg ggc cgg ctc cgc atg cgc aag aac tca ggc ctc 2366 ThrAsp Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu 750 755 760765 aag gta att ctg gga att cta ctt cct cct tca att ctc agc ttg gag 2414Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu 770 775780 ttc aag aac aaa gac gac atg ccc tat atg tct cag gcc cag gaa atc 2462Phe Lys Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile 785 790795 cac ctc caa gag aag gag gca gaa gaa cca gag aag ccc aca aag gaa 2510His Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu 800 805810 aaa gag gaa gag gac atg gag ctc aca gca atg ttg gga cga aac aac 2558Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn Asn 815 820825 ggg gag tcc tcc agg aag aag gat gaa gag gaa gtt cag agc aag cac 2606Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His 830 835840 845 cgg tta atc ccc ctc ggc aga aaa atc tat gaa ttc tac aat gca ccc2654 Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro 850855 860 atc gtg aag ttc tgg ttc tac aca ctg gcg tat atc gga tac ctg atg2702 Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met 865870 875 ctc ttc aac tat atc gtg tta gtg aag atg gaa cgc tgg ccg tcc acc2750 Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser Thr 880885 890 cag gaa tgg atc gta atc tcc tat att ttc acc ctg gga ata gaa aag2798 Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys 895900 905 atg aga gag att ctg atg tca gag cca ggg aag ttg cta cag aaa gtg2846 Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys Val 910915 920 925 aag gta tgg ctg cag gag tac tgg aat gtc acg gac ctc atc gccatc 2894 Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp Leu Ile Ala Ile930 935 940 ctt ctg ttt tct gtc gga atg atc ctt cgt ctc caa gac cag cccttc 2942 Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln Asp Gln Pro Phe945 950 955 agg agt gac ggg agg gtc atc tac tgc gtg aac atc att tac tggtat 2990 Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr960 965 970 atc cgt ctc cta gac atc ttc ggc gtg aac aag tat ttg ggc ccgtat 3038 Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr975 980 985 gta atg atg att gga aaa atg atg ata gac atg atg tac ttt gtcatc 3086 Val Met Met Ile Gly Lys Met Met Ile Asp Met Met Tyr Phe Val Ile990 995 1000 1005 att atg ctg gtg gtt ctg atg agc ttt ggg gtc gcc aggcaa gcc 3131 Ile Met Leu Val Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala1010 1015 1020 atc ctt ttt ccc aat gag gag cca tca tgg aaa ctg gcc aagaac 3176 Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn1025 1030 1035 atc ttc tac atg ccc tat tgg atg att tat ggg gaa gtg tttgcg 3221 Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala1040 1045 1050 gac cag ata gac cct ccc tgt gga cag aat gag acc cga gaggat 3266 Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp1055 1060 1065 ggt aaa ata atc cag ctg cct ccc tgc aag aca gga gct tggatc 3311 Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile1070 1075 1080 gtg ccg gcc atc atg gcc tgc tac ctc tta gtg gca aac atcttg 3356 Val Pro Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu1085 1090 1095 ctg gtc aac ctc ctc att gct gtc ttt aac aat aca ttt tttgaa 3401 Leu Val Asn Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu1100 1105 1110 gta aaa tcg ata tcc aac caa gtc tgg aag ttt cag agg tatcag 3446 Val Lys Ser Ile Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln1115 1120 1125 ctc atc atg act ttc cat gaa agg cca gtt ctg ccc cca ccactg 3491 Leu Ile Met Thr Phe His Glu Arg Pro Val Leu Pro Pro Pro Leu1130 1135 1140 atc atc ttc agc cac atg acc atg ata ttc cag cac ctg tgctgc 3536 Ile Ile Phe Ser His Met Thr Met Ile Phe Gln His Leu Cys Cys1145 1150 1155 cga tgg agg aaa cac gag agc gac ccg gat gaa agg gac tacggc 3581 Arg Trp Arg Lys His Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly1160 1165 1170 ctg aaa ctc ttc ata acc gat gat gag ctc aag aaa gta catgac 3626 Leu Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys Lys Val His Asp1175 1180 1185 ttt gaa gag caa tgc ata gaa gaa tac ttc aga gaa aag gatgat 3671 Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys Asp Asp1190 1195 1200 cgg ttc aac tca tct aat gat gag agg ata cgg gtg act tcagaa 3716 Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg Val Thr Ser Glu1205 1210 1215 agg gtg gag aac atg tct atg cgg ctg gag gaa gtc aac gagaga 3761 Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu Val Asn Glu Arg1220 1225 1230 gag cac tcc atg aag gct tca ctc cag acc gtg gac atc cggctg 3806 Glu His Ser Met Lys Ala Ser Leu Gln Thr Val Asp Ile Arg Leu1235 1240 1245 gcg cag ctg gaa gac ctt atc ggg cgc atg gcc acg gcc ctggag 3851 Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala Leu Glu1250 1255 1260 cgc ctg aca ggt ctg gag cgg gcc gag tcc aac aaa atc cgctcg 3896 Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg Ser1265 1270 1275 agg acc tcg tca gac tgc acg gac gcc gcc tac att gtc cgtcag 3941 Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln1280 1285 1290 agc agc ttc aac agc cag gaa ggg aac acc ttc aag ctc caagag 3986 Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu1295 1300 1305 agt ata gac cct gca ggt gag gag acc atg tcc cca act tctcca 4031 Ser Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr Ser Pro1310 1315 1320 acc tta atg ccc cgt atg cga agc cat tct ttc tat tca gtcaat 4076 Thr Leu Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser Val Asn1325 1330 1335 atg aaa gac aaa ggt ggt ata gaa aag ttg gaa agt att tttaaa 4121 Met Lys Asp Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys1340 1345 1350 gaa agg tcc ctg agc cta cac cgg gct act agt tcc cac tctgta 4166 Glu Arg Ser Leu Ser Leu His Arg Ala Thr Ser Ser His Ser Val1355 1360 1365 gca aaa gaa ccc aaa gct cct gca gcc cct gcc aac acc ttggcc 4211 Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala1370 1375 1380 att gtt cct gat tcc aga aga cca tca tcg tgt ata gac atctat 4256 Ile Val Pro Asp Ser Arg Arg Pro Ser Ser Cys Ile Asp Ile Tyr1385 1390 1395 gtc tct gct atg gat gag ctc cac tgt gat ata gac cct ctggac 4301 Val Ser Ala Met Asp Glu Leu His Cys Asp Ile Asp Pro Leu Asp1400 1405 1410 aat tcc gtg aac atc ctt ggg cta ggc gag cca agc ttt tcaact 4346 Asn Ser Val Asn Ile Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr1415 1420 1425 cca gta cct tcc aca gcc cct tca agt agt gcc tat gca acactt 4391 Pro Val Pro Ser Thr Ala Pro Ser Ser Ser Ala Tyr Ala Thr Leu1430 1435 1440 gca ccc aca gac aga cct cca agc cgg agc att gat ttt gaggac 4436 Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe Glu Asp1445 1450 1455 atc acc tcc atg gac act aga tct ttt tct tca gac tac acccac 4481 Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr Thr His1460 1465 1470 ctc cca gaa tgc caa aac ccc tgg gac tca gag cct ccg atgtac 4526 Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu Pro Pro Met Tyr1475 1480 1485 cac acc att gag cgt tcc aaa agt agc cgc tac cta gcc accaca 4571 His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr1490 1495 1500 ccc ttt ctt cta gaa gag gct ccc att gtg aaa tct cat agcttt 4616 Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser Phe1505 1510 1515 atg ttt tcc ccc tca agg agc tat tat gcc aac ttt ggg gtgcct 4661 Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro1520 1525 1530 gta aaa aca gca gaa tac aca agt att aca gac tgt att gacaca 4706 Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr1535 1540 1545 agg tgt gtc aat gcc cct caa gca att gcg gac aga gct gccttc 4751 Arg Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala Ala Phe1550 1555 1560 cct gga ggt ctt gga gac aaa gtg gag gac tta act tgc tgccat 4796 Pro Gly Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys Cys His1565 1570 1575 cca gag cga gaa gca gaa ctg agt cac ccc agc tct gac agtgag 4841 Pro Glu Arg Glu Ala Glu Leu Ser His Pro Ser Ser Asp Ser Glu1580 1585 1590 gag aat gag gcc aaa ggc cgc aga gcc acc att gca ata tcctcc 4886 Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser1595 1600 1605 cag gag ggt gat aac tca gag aga acc ctg tcc aac aac atcact 4931 Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr1610 1615 1620 gtt ccc aag ata gag cgc gcc aac agc tac tcg gca gag gagcca 4976 Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr Ser Ala Glu Glu Pro1625 1630 1635 agt gcg cca tat gca cac acc agg aag agc ttc tcc atc agtgac 5021 Ser Ala Pro Tyr Ala His Thr Arg Lys Ser Phe Ser Ile Ser Asp1640 1645 1650 aaa ctc gac agg cag cgg aac aca gca agc ctg caa aat cccttc 5066 Lys Leu Asp Arg Gln Arg Asn Thr Ala Ser Leu Gln Asn Pro Phe1655 1660 1665 cag aga agc aag tcc tcc aag ccg gag ggc cga ggg gac agcctg 5111 Gln Arg Ser Lys Ser Ser Lys Pro Glu Gly Arg Gly Asp Ser Leu1670 1675 1680 tcc atg agg aga ctg tcc aga aca tcg gct ttc caa agc tttgaa 5156 Ser Met Arg Arg Leu Ser Arg Thr Ser Ala Phe Gln Ser Phe Glu1685 1690 1695 agc aag cac acc taa 5171 Ser Lys His Thr 8 1699 PRT Homosapiens 8 Met Gly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg Gly CysSer 1 5 10 15 Asp Arg Glu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg SerAla Ser 20 25 30 Arg Gly Arg Phe Ala Glu Ser Trp Lys Arg Leu Ser Ser LysGln Gly 35 40 45 Ser Thr Lys Arg Ser Gly Leu Pro Ser Gln Gln Thr Pro AlaGln Lys 50 55 60 Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val HisIle Ile 65 70 75 80 Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly ArgLeu Ile Gly 85 90 95 Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu GlnAsn Glu Lys 100 105 110 Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln SerGlu Lys Trp Ser 115 120 125 Ile Ser Lys His Thr Gln Leu Ser Pro Thr AspAla Phe Gly Thr Ile 130 135 140 Glu Phe Gln Gly Gly Gly His Ser Asn LysAla Met Tyr Val Arg Val 145 150 155 160 Ser Phe Asp Thr Lys Pro Asp LeuLeu Leu His Leu Met Thr Lys Glu 165 170 175 Trp Gln Leu Glu Leu Pro LysLeu Leu Ile Ser Val His Gly Gly Leu 180 185 190 Gln Asn Phe Glu Leu GlnPro Lys Leu Lys Gln Val Phe Gly Lys Gly 195 200 205 Leu Ile Lys Ala AlaMet Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly 210 215 220 Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu Lys Asp His 225 230 235 240 Ala SerLys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro Trp 245 250 255 GlyIle Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val Arg 260 265 270Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu Asn 275 280285 Ser Met His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr Gly Lys 290295 300 Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys His Ile Ser305 310 315 320 Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro ValVal Ala 325 330 335 Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser Ile ValLeu Glu Tyr 340 345 350 Leu Arg Asp Thr Pro Pro Val Pro Val Val Val CysAsp Gly Ser Gly 355 360 365 Arg Ala Ser Asp Ile Leu Ala Phe Gly His LysTyr Ser Glu Glu Gly 370 375 380 Gly Leu Ile Asn Glu Ser Leu Arg Asp GlnLeu Leu Val Thr Ile Gln 385 390 395 400 Lys Thr Phe Thr Tyr Thr Arg ThrGln Ala Gln His Leu Phe Ile Ile 405 410 415 Leu Met Glu Cys Met Lys LysLys Glu Leu Ile Thr Val Phe Arg Met 420 425 430 Gly Ser Glu Gly His GlnAsp Ile Asp Leu Ala Ile Leu Thr Ala Leu 435 440 445 Leu Lys Gly Ala AsnAla Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu 450 455 460 Ala Trp Asn ArgVal Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly 465 470 475 480 Gln GlnTrp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu 485 490 495 ValLeu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val 500 505 510Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr Asn 515 520525 Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp Val 530535 540 Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile Asp Ile545 550 555 560 Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg CysAsn Tyr 565 570 575 Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn Leu PheGly Pro Lys 580 585 590 Arg Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys ThrThr Lys Lys Arg 595 600 605 Glu Glu Glu Val Asp Ile Asp Leu Asp Asp ProGlu Ile Asn His Phe 610 615 620 Pro Phe Pro Phe His Glu Leu Met Val TrpAla Val Leu Met Lys Arg 625 630 635 640 Gln Lys Met Ala Leu Phe Phe TrpGln His Gly Glu Glu Ala Met Ala 645 650 655 Lys Ala Leu Val Ala Cys LysLeu Cys Lys Ala Met Ala His Glu Ala 660 665 670 Ser Glu Asn Asp Met ValAsp Asp Ile Ser Gln Glu Leu Asn His Asn 675 680 685 Ser Arg Asp Phe GlyGln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr 690 695 700 Lys Gln Asp GluGln Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys 705 710 715 720 Asn TrpSer Asn Ala Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His 725 730 735 ArgAsp Phe Ile Ala His Thr Cys Ser Gln Met Leu Leu Thr Asp Met 740 745 750Trp Met Gly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile 755 760765 Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn 770775 780 Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln785 790 795 800 Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu LysGlu Glu 805 810 815 Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn AsnGly Glu Ser 820 825 830 Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser LysHis Arg Leu Ile 835 840 845 Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr AsnAla Pro Ile Val Lys 850 855 860 Phe Trp Phe Tyr Thr Leu Ala Tyr Ile GlyTyr Leu Met Leu Phe Asn 865 870 875 880 Tyr Ile Val Leu Val Lys Met GluArg Trp Pro Ser Thr Gln Glu Trp 885 890 895 Ile Val Ile Ser Tyr Ile PheThr Leu Gly Ile Glu Lys Met Arg Glu 900 905 910 Ile Leu Met Ser Glu ProGly Lys Leu Leu Gln Lys Val Lys Val Trp 915 920 925 Leu Gln Glu Tyr TrpAsn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe 930 935 940 Ser Val Gly MetIle Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp 945 950 955 960 Gly ArgVal Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu 965 970 975 LeuAsp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met 980 985 990Ile Gly Lys Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu 995 10001005 Val Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe 10101015 1020 Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr1025 1030 1035 Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp GlnIle 1040 1045 1050 Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp GlyLys Ile 1055 1060 1065 Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp IleVal Pro Ala 1070 1075 1080 Ile Met Ala Cys Tyr Leu Leu Val Ala Asn IleLeu Leu Val Asn 1085 1090 1095 Leu Leu Ile Ala Val Phe Asn Asn Thr PhePhe Glu Val Lys Ser 1100 1105 1110 Ile Ser Asn Gln Val Trp Lys Phe GlnArg Tyr Gln Leu Ile Met 1115 1120 1125 Thr Phe His Glu Arg Pro Val LeuPro Pro Pro Leu Ile Ile Phe 1130 1135 1140 Ser His Met Thr Met Ile PheGln His Leu Cys Cys Arg Trp Arg 1145 1150 1155 Lys His Glu Ser Asp ProAsp Glu Arg Asp Tyr Gly Leu Lys Leu 1160 1165 1170 Phe Ile Thr Asp AspGlu Leu Lys Lys Val His Asp Phe Glu Glu 1175 1180 1185 Gln Cys Ile GluGlu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn 1190 1195 1200 Ser Ser AsnAsp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu 1205 1210 1215 Asn MetSer Met Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser 1220 1225 1230 MetLys Ala Ser Leu Gln Thr Val Asp Ile Arg Leu Ala Gln Leu 1235 1240 1245Glu Asp Leu Ile Gly Arg Met Ala Thr Ala Leu Glu Arg Leu Thr 1250 12551260 Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser 12651270 1275 Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe1280 1285 1290 Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu Ser IleAsp 1295 1300 1305 Pro Ala Gly Glu Glu Thr Met Ser Pro Thr Ser Pro ThrLeu Met 1310 1315 1320 Pro Arg Met Arg Ser His Ser Phe Tyr Ser Val AsnMet Lys Asp 1325 1330 1335 Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile PheLys Glu Arg Ser 1340 1345 1350 Leu Ser Leu His Arg Ala Thr Ser Ser HisSer Val Ala Lys Glu 1355 1360 1365 Pro Lys Ala Pro Ala Ala Pro Ala AsnThr Leu Ala Ile Val Pro 1370 1375 1380 Asp Ser Arg Arg Pro Ser Ser CysIle Asp Ile Tyr Val Ser Ala 1385 1390 1395 Met Asp Glu Leu His Cys AspIle Asp Pro Leu Asp Asn Ser Val 1400 1405 1410 Asn Ile Leu Gly Leu GlyGlu Pro Ser Phe Ser Thr Pro Val Pro 1415 1420 1425 Ser Thr Ala Pro SerSer Ser Ala Tyr Ala Thr Leu Ala Pro Thr 1430 1435 1440 Asp Arg Pro ProSer Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser 1445 1450 1455 Met Asp ThrArg Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu 1460 1465 1470 Cys GlnAsn Pro Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile 1475 1480 1485 GluArg Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu 1490 1495 1500Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser Phe Met Phe Ser 1505 15101515 Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr 15201525 1530 Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val1535 1540 1545 Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala Ala Phe Pro GlyGly 1550 1555 1560 Leu Gly Asp Lys Val Glu Asp Leu Thr Cys Cys His ProGlu Arg 1565 1570 1575 Glu Ala Glu Leu Ser His Pro Ser Ser Asp Ser GluGlu Asn Glu 1580 1585 1590 Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile SerSer Gln Glu Gly 1595 1600 1605 Asp Asn Ser Glu Arg Thr Leu Ser Asn AsnIle Thr Val Pro Lys 1610 1615 1620 Ile Glu Arg Ala Asn Ser Tyr Ser AlaGlu Glu Pro Ser Ala Pro 1625 1630 1635 Tyr Ala His Thr Arg Lys Ser PheSer Ile Ser Asp Lys Leu Asp 1640 1645 1650 Arg Gln Arg Asn Thr Ala SerLeu Gln Asn Pro Phe Gln Arg Ser 1655 1660 1665 Lys Ser Ser Lys Pro GluGly Arg Gly Asp Ser Leu Ser Met Arg 1670 1675 1680 Arg Leu Ser Arg ThrSer Ala Phe Gln Ser Phe Glu Ser Lys His 1685 1690 1695 Thr 9 5207 DNAHomo sapiens CDS (72)..(5204) 9 cgcccgcggc gaggagccag cgagagcgctcggcgctggg ctgtttcccg gccgagggag 60 gcgaacttct c atg ggg aag aag tgg agggat gcg gcg gaa atg gag cgg 110 Met Gly Lys Lys Trp Arg Asp Ala Ala GluMet Glu Arg 1 5 10 ggc tgc tcc gac cgc gag gac aac gcg gag agc cgc agacgc agc cgg 158 Gly Cys Ser Asp Arg Glu Asp Asn Ala Glu Ser Arg Arg ArgSer Arg 15 20 25 agc gcc agc cgg ggc agg ttt gcc gag tcg tgg aaa agg ttaagt tcc 206 Ser Ala Ser Arg Gly Arg Phe Ala Glu Ser Trp Lys Arg Leu SerSer 30 35 40 45 aag cag ggg tcc acc aaa cgc tcg gga ctc ccg tcg cag cagacg ccg 254 Lys Gln Gly Ser Thr Lys Arg Ser Gly Leu Pro Ser Gln Gln ThrPro 50 55 60 gct cag aaa tcc tgg ata gaa aga gca ttt tat aaa aga gaa tgtgtc 302 Ala Gln Lys Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val65 70 75 cac atc ata ccc agc acc aaa gac ccc cat agg tgt tgc tgt ggg cgt350 His Ile Ile Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly Arg 8085 90 ctg ata ggc cag cat gtt ggc ctc acc ccc agt atc tcc gtg ctt cag398 Leu Ile Gly Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu Gln 95100 105 aat gag aaa aat gaa agt cgc ctc tcc cga aat gac atc cag tct gaa446 Asn Glu Lys Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu 110115 120 125 aag tgg tcc atc agc aaa cac act caa ctc agc cct acg gat gctttt 494 Lys Trp Ser Ile Ser Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe130 135 140 ggg acc att gag ttc caa gga ggt ggc cat tcc aac aaa gcc atgtat 542 Gly Thr Ile Glu Phe Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr145 150 155 gtg cga gta tct ttt gat aca aaa cct gat ctc ctc tta cac ctgatg 590 Val Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met160 165 170 acc aag gaa tgg cag ttg gag ctt ccc aag ctt ctc atc tct gtccat 638 Thr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His175 180 185 ggg ggc ctg cag aac ttt gaa ctc cag cca aaa ctc aag caa gtcttt 686 Gly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln Val Phe190 195 200 205 ggg aaa ggg ctc atc aaa gca gca atg aca act gga gcg tggata ttc 734 Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp IlePhe 210 215 220 act gga ggg gtt aac aca ggt gtt att cgt cat gtt ggc gatgcc ttg 782 Thr Gly Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp AlaLeu 225 230 235 aag gat cat gcc tct aag tct cga gga aag ata tgc acc ataggt att 830 Lys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile GlyIle 240 245 250 gcc ccc tgg gga att gtg gaa aac cag gag gac ctc att ggaaga gat 878 Ala Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly ArgAsp 255 260 265 gtt gtc cgg cca tac cag acc atg tcc aat ccc atg agc aagctc act 926 Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys LeuThr 270 275 280 285 gtt ctc aac agc atg cat tcc cac ttc att ctg gct gacaac ggg acc 974 Val Leu Asn Ser Met His Ser His Phe Ile Leu Ala Asp AsnGly Thr 290 295 300 act gga aaa tat gga gca gag gtg aaa ctt cga aga caactg gaa aag 1022 Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln LeuGlu Lys 305 310 315 cat att tca ctc cag aag ata aac aca aga atc ggt caaggt gtt cct 1070 His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln GlyVal Pro 320 325 330 gtg gtg gca ctc ata gtg gaa gga gga ccc aat gtg atctcg att gtt 1118 Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile SerIle Val 335 340 345 ttg gag tac ctt cga gac acc cct ccc gtg cca gtg gttgtc tgt gat 1166 Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val ValCys Asp 350 355 360 365 ggg agt gga cgg gca tcg gac atc ctg gcc ttt gggcat aaa tac tca 1214 Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly HisLys Tyr Ser 370 375 380 gaa gaa ggc gga ctg ata aat gaa tct ttg agg gaccag ctg ttg gtg 1262 Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu Arg Asp GlnLeu Leu Val 385 390 395 act ata cag aag act ttc aca tac act cga acc caagct cag cat ctg 1310 Thr Ile Gln Lys Thr Phe Thr Tyr Thr Arg Thr Gln AlaGln His Leu 400 405 410 ttc atc atc ctc atg gag tgc atg aag aag aag gaattg att acg gta 1358 Phe Ile Ile Leu Met Glu Cys Met Lys Lys Lys Glu LeuIle Thr Val 415 420 425 ttt cgg atg gga tca gaa gga cac cag gac att gatttg gct atc ctg 1406 Phe Arg Met Gly Ser Glu Gly His Gln Asp Ile Asp LeuAla Ile Leu 430 435 440 445 aca gct tta ctc aaa gga gcc aat gcc tcg gcccca gac caa ctg agc 1454 Thr Ala Leu Leu Lys Gly Ala Asn Ala Ser Ala ProAsp Gln Leu Ser 450 455 460 tta gct tta gcc tgg aac aga gtc gac atc gctcgc agc cag atc ttt 1502 Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala ArgSer Gln Ile Phe 465 470 475 att tac ggg caa cag tgg ccg gtg gga tct ctggag caa gcc atg ttg 1550 Ile Tyr Gly Gln Gln Trp Pro Val Gly Ser Leu GluGln Ala Met Leu 480 485 490 gat gcc tta gtt ctg gac aga gtg gat ttt gtgaaa tta ctc ata gag 1598 Asp Ala Leu Val Leu Asp Arg Val Asp Phe Val LysLeu Leu Ile Glu 495 500 505 aat gga gta agc atg cac cgt ttt ctc acc atctcc aga cta gag gaa 1646 Asn Gly Val Ser Met His Arg Phe Leu Thr Ile SerArg Leu Glu Glu 510 515 520 525 ttg tac aat acg aga cat ggg ccc tca aataca ttg tac cac ttg gtc 1694 Leu Tyr Asn Thr Arg His Gly Pro Ser Asn ThrLeu Tyr His Leu Val 530 535 540 agg gat gtc aaa aag cga gag tat cca ggtttc ggt tgg atc tat ttt 1742 Arg Asp Val Lys Lys Arg Glu Tyr Pro Gly PheGly Trp Ile Tyr Phe 545 550 555 aag ggg aac ctg ccc cca gac tac aga atcagc ctg att gac atc ggc 1790 Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile SerLeu Ile Asp Ile Gly 560 565 570 ctg gtg atc gag tac ctg atg ggc ggg gcttat cgc tgc aac tac acg 1838 Leu Val Ile Glu Tyr Leu Met Gly Gly Ala TyrArg Cys Asn Tyr Thr 575 580 585 cgc aag cgc ttc cgg acc ctc tac cac aacctc ttc ggc ccc aag agg 1886 Arg Lys Arg Phe Arg Thr Leu Tyr His Asn LeuPhe Gly Pro Lys Arg 590 595 600 605 gat gat att ccc ttg agg cga gga agaaag aca acc aag aaa cgt gaa 1934 Asp Asp Ile Pro Leu Arg Arg Gly Arg LysThr Thr Lys Lys Arg Glu 610 615 620 gaa gag gtg gac att gac ttg gat gatcct gag atc aac cac ttc ccc 1982 Glu Glu Val Asp Ile Asp Leu Asp Asp ProGlu Ile Asn His Phe Pro 625 630 635 ttc cct ttc cat gag ctc atg gtg tgggct gtt ctc atg aag cgg cag 2030 Phe Pro Phe His Glu Leu Met Val Trp AlaVal Leu Met Lys Arg Gln 640 645 650 aag atg gcc ctg ttc ttc tgg cag cacggt gag gag gcc atg gcc aag 2078 Lys Met Ala Leu Phe Phe Trp Gln His GlyGlu Glu Ala Met Ala Lys 655 660 665 gcc ctg gtg gcc tgc aag ctc tgc aaagcc atg gct cat gag gcc tct 2126 Ala Leu Val Ala Cys Lys Leu Cys Lys AlaMet Ala His Glu Ala Ser 670 675 680 685 gag aac gac atg gtt gac gac atttcc cag gag ctg aat cac aat tcc 2174 Glu Asn Asp Met Val Asp Asp Ile SerGln Glu Leu Asn His Asn Ser 690 695 700 aga gac ttt ggc cag ctg gct gtggag ctc ctg gac cag tcc tac aag 2222 Arg Asp Phe Gly Gln Leu Ala Val GluLeu Leu Asp Gln Ser Tyr Lys 705 710 715 cag gac gaa cag ctg gcc atg aaactg ctg acg tat gag ctg aag aac 2270 Gln Asp Glu Gln Leu Ala Met Lys LeuLeu Thr Tyr Glu Leu Lys Asn 720 725 730 tgg agc aac gcc acg tgc ctg cagctt gcc gtg gct gcc aaa cac cgc 2318 Trp Ser Asn Ala Thr Cys Leu Gln LeuAla Val Ala Ala Lys His Arg 735 740 745 gac ttc atc gcg cac acg tgc agccag atg ctg ctc acc gac atg tgg 2366 Asp Phe Ile Ala His Thr Cys Ser GlnMet Leu Leu Thr Asp Met Trp 750 755 760 765 atg ggc cgg ctc cgc atg cgcaag aac tca ggc ctc aag gta att ctg 2414 Met Gly Arg Leu Arg Met Arg LysAsn Ser Gly Leu Lys Val Ile Leu 770 775 780 gga att cta ctt cct cct tcaatt ctc agc ttg gag ttc aag aac aaa 2462 Gly Ile Leu Leu Pro Pro Ser IleLeu Ser Leu Glu Phe Lys Asn Lys 785 790 795 gac gac atg ccc tat atg tctcag gcc cag gaa atc cac ctc caa gag 2510 Asp Asp Met Pro Tyr Met Ser GlnAla Gln Glu Ile His Leu Gln Glu 800 805 810 aag gag gca gaa gaa cca gagaag ccc aca aag gaa aaa gag gaa gag 2558 Lys Glu Ala Glu Glu Pro Glu LysPro Thr Lys Glu Lys Glu Glu Glu 815 820 825 gac atg gag ctc aca gca atgttg gga cga aac aac ggg gag tcc tcc 2606 Asp Met Glu Leu Thr Ala Met LeuGly Arg Asn Asn Gly Glu Ser Ser 830 835 840 845 agg aag aag gat gaa gaggaa gtt cag agc aag cac cgg tta atc ccc 2654 Arg Lys Lys Asp Glu Glu GluVal Gln Ser Lys His Arg Leu Ile Pro 850 855 860 ctc ggc aga aaa atc tatgaa ttc tac aat gca ccc atc gtg aag ttc 2702 Leu Gly Arg Lys Ile Tyr GluPhe Tyr Asn Ala Pro Ile Val Lys Phe 865 870 875 tgg ttc tac aca ctg gcgtat atc gga tac ctg atg ctc ttc aac tat 2750 Trp Phe Tyr Thr Leu Ala TyrIle Gly Tyr Leu Met Leu Phe Asn Tyr 880 885 890 atc gtg tta gtg aag atggaa cgc tgg ccg tcc acc cag gaa tgg atc 2798 Ile Val Leu Val Lys Met GluArg Trp Pro Ser Thr Gln Glu Trp Ile 895 900 905 gta atc tcc tat att ttcacc ctg gga ata gaa aag atg aga gag att 2846 Val Ile Ser Tyr Ile Phe ThrLeu Gly Ile Glu Lys Met Arg Glu Ile 910 915 920 925 ctg atg tca gag ccaggg aag ttg cta cag aaa gtg aag gta tgg ctg 2894 Leu Met Ser Glu Pro GlyLys Leu Leu Gln Lys Val Lys Val Trp Leu 930 935 940 cag gag tac tgg aatgtc acg gac ctc atc gcc atc ctt ctg ttt tct 2942 Gln Glu Tyr Trp Asn ValThr Asp Leu Ile Ala Ile Leu Leu Phe Ser 945 950 955 gtc gga atg atc cttcgt ctc caa gac cag ccc ttc agg agt gac ggg 2990 Val Gly Met Ile Leu ArgLeu Gln Asp Gln Pro Phe Arg Ser Asp Gly 960 965 970 agg gtc atc tac tgcgtg aac atc att tac tgg tat atc cgt ctc cta 3038 Arg Val Ile Tyr Cys ValAsn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 975 980 985 gac atc ttc ggc gtgaac aag tat ttg ggc ccg tat gta atg atg att 3086 Asp Ile Phe Gly Val AsnLys Tyr Leu Gly Pro Tyr Val Met Met Ile 990 995 1000 1005 gga aaa atgatg ata gac atg atg tac ttt gtc atc att atg ctg 3131 Gly Lys Met Met IleAsp Met Met Tyr Phe Val Ile Ile Met Leu 1010 1015 1020 gtg gtt ctg atgagc ttt ggg gtc gcc agg caa gcc atc ctt ttt 3176 Val Val Leu Met Ser PheGly Val Ala Arg Gln Ala Ile Leu Phe 1025 1030 1035 ccc aat gag gag ccatca tgg aaa ctg gcc aag aac atc ttc tac 3221 Pro Asn Glu Glu Pro Ser TrpLys Leu Ala Lys Asn Ile Phe Tyr 1040 1045 1050 atg ccc tat tgg atg atttat ggg gaa gtg ttt gcg gac cag ata 3266 Met Pro Tyr Trp Met Ile Tyr GlyGlu Val Phe Ala Asp Gln Ile 1055 1060 1065 gac cct ccc tgt gga cag aatgag acc cga gag gat ggt aaa ata 3311 Asp Pro Pro Cys Gly Gln Asn Glu ThrArg Glu Asp Gly Lys Ile 1070 1075 1080 atc cag ctg cct ccc tgc aag acagga gct tgg atc gtg ccg gcc 3356 Ile Gln Leu Pro Pro Cys Lys Thr Gly AlaTrp Ile Val Pro Ala 1085 1090 1095 atc atg gcc tgc tac ctc tta gtg gcaaac atc ttg ctg gtc aac 3401 Ile Met Ala Cys Tyr Leu Leu Val Ala Asn IleLeu Leu Val Asn 1100 1105 1110 ctc ctc att gct gtc ttt aac aat aca tttttt gaa gta aaa tcg 3446 Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe GluVal Lys Ser 1115 1120 1125 ata tcc aac caa gtc tgg aag ttt cag agg tatcag ctc atc atg 3491 Ile Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln LeuIle Met 1130 1135 1140 act ttc cat gaa agg cca gtt ctg ccc cca cca ctgatc atc ttc 3536 Thr Phe His Glu Arg Pro Val Leu Pro Pro Pro Leu Ile IlePhe 1145 1150 1155 agc cac atg acc atg ata ttc cag cac ctg tgc tgc cgatgg agg 3581 Ser His Met Thr Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg1160 1165 1170 aaa cac gag agc gac ccg gat gaa agg gac tac ggc ctg aaactc 3626 Lys His Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu1175 1180 1185 ttc ata acc gat gat gag ctc aag aaa gta cat gac ttt gaagag 3671 Phe Ile Thr Asp Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu1190 1195 1200 caa tgc ata gaa gaa tac ttc aga gaa aag gat gat cgg ttcaac 3716 Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn1205 1210 1215 tca tct aat gat gag agg ata cgg gtg act tca gaa agg gtggag 3761 Ser Ser Asn Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu1220 1225 1230 aac atg tct atg cgg ctg gag gaa gtc aac gag aga gag cactcc 3806 Asn Met Ser Met Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser1235 1240 1245 atg aag gct tca ctc cag acc gtg gac atc cgg ctg gcg cagctg 3851 Met Lys Ala Ser Leu Gln Thr Val Asp Ile Arg Leu Ala Gln Leu1250 1255 1260 gaa gac ctt atc ggg cgc atg gcc acg gcc ctg gag cgc ctgaca 3896 Glu Asp Leu Ile Gly Arg Met Ala Thr Ala Leu Glu Arg Leu Thr1265 1270 1275 ggt ctg gag cgg gcc gag tcc aac aaa atc cgc tcg agg acctcg 3941 Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser1280 1285 1290 tca gac tgc acg gac gcc gcc tac att gtc cgt cag agc agcttc 3986 Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe1295 1300 1305 aac agc cag gaa ggg aac acc ttc aag ctc caa gag agt atagac 4031 Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp1310 1315 1320 cct gca ggt gag gag acc atg tcc cca act tct cca acc ttaatg 4076 Pro Ala Gly Glu Glu Thr Met Ser Pro Thr Ser Pro Thr Leu Met1325 1330 1335 ccc cgt atg cga agc cat tct ttc tat tca gtc aat atg aaagac 4121 Pro Arg Met Arg Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp1340 1345 1350 aaa ggt ggt ata gaa aag ttg gaa agt att ttt aaa gaa aggtcc 4166 Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser1355 1360 1365 ctg agc cta cac cgg gct act agt tcc cac tct gta gca aaagaa 4211 Leu Ser Leu His Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu1370 1375 1380 ccc aaa gct cct gca gcc cct gcc aac acc ttg gcc att gttcct 4256 Pro Lys Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro1385 1390 1395 gat tcc aga aga cca tca tcg tgt ata gac atc tat gtc tctgct 4301 Asp Ser Arg Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala1400 1405 1410 atg gat gag ctc cac tgt gat ata gac cct ctg gac aat tccgtg 4346 Met Asp Glu Leu His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val1415 1420 1425 aac atc ctt ggg cta ggc gag cca agc ttt tca act cca gtacct 4391 Asn Ile Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro1430 1435 1440 tcc aca gcc cct tca agt agt gcc tat gca aca ctt gca cccaca 4436 Ser Thr Ala Pro Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr1445 1450 1455 gac aga cct cca agc cgg agc att gat ttt gag gac atc acctcc 4481 Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser1460 1465 1470 atg gac act aga tct ttt tct tca gac tac acc cac ctc ccagaa 4526 Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu1475 1480 1485 tgc caa aac ccc tgg gac tca gag cct ccg atg tac cac accatt 4571 Cys Gln Asn Pro Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile1490 1495 1500 gag cgt tcc aaa agt agc cgc tac cta gcc acc aca ccc tttctt 4616 Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu1505 1510 1515 cta gaa gag gct ccc att gtg aaa tct cat agc ttt atg ttttcc 4661 Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser Phe Met Phe Ser1520 1525 1530 ccc tca agg agc tat tat gcc aac ttt ggg gtg cct gta aaaaca 4706 Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr1535 1540 1545 gca gaa tac aca agt att aca gac tgt att gac aca agg tgtgtc 4751 Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val1550 1555 1560 aat gcc cct caa gca att gcg gac aga gct gcc ttc cct ggaggt 4796 Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly1565 1570 1575 ctt gga gac aaa gtg gag gac tta act tgc tgc cat cca gagcga 4841 Leu Gly Asp Lys Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg1580 1585 1590 gaa gca gaa ctg agt cac ccc agc tct gac agt gag gag aatgag 4886 Glu Ala Glu Leu Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu1595 1600 1605 gcc aaa ggc cgc aga gcc acc att gca ata tcc tcc cag gagggt 4931 Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly1610 1615 1620 gat aac tca gag aga acc ctg tcc aac aac atc act gtt cccaag 4976 Asp Asn Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys1625 1630 1635 ata gag cgc gcc aac agc tac tcg gca gag gag cca agt gcgcca 5021 Ile Glu Arg Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro1640 1645 1650 tat gca cac acc agg aag agc ttc tcc atc agt gac aaa ctcgac 5066 Tyr Ala His Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp1655 1660 1665 agg cag cgg aac aca gca agc ctg caa aat ccc ttc cag agaagc 5111 Arg Gln Arg Asn Thr Ala Ser Leu Gln Asn Pro Phe Gln Arg Ser1670 1675 1680 aag tcc tcc aag ccg gag ggc cga ggg gac agc ctg tcc atgagg 5156 Lys Ser Ser Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg1685 1690 1695 aga ctg tcc aga aca tcg gct ttc caa agc ttt gaa agc aagcac 5201 Arg Leu Ser Arg Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His1700 1705 1710 acc taa 5207 Thr 10 1711 PRT Homo sapiens 10 Met Gly LysLys Trp Arg Asp Ala Ala Glu Met Glu Arg Gly Cys Ser 1 5 10 15 Asp ArgGlu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg Ser Ala Ser 20 25 30 Arg GlyArg Phe Ala Glu Ser Trp Lys Arg Leu Ser Ser Lys Gln Gly 35 40 45 Ser ThrLys Arg Ser Gly Leu Pro Ser Gln Gln Thr Pro Ala Gln Lys 50 55 60 Ser TrpIle Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val His Ile Ile 65 70 75 80 ProSer Thr Lys Asp Pro His Arg Cys Cys Cys Gly Arg Leu Ile Gly 85 90 95 GlnHis Val Gly Leu Thr Pro Ser Ile Ser Val Leu Gln Asn Glu Lys 100 105 110Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu Lys Trp Ser 115 120125 Ile Ser Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe Gly Thr Ile 130135 140 Glu Phe Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr Val Arg Val145 150 155 160 Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met ThrLys Glu 165 170 175 Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val HisGly Gly Leu 180 185 190 Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln ValPhe Gly Lys Gly 195 200 205 Leu Ile Lys Ala Ala Met Thr Thr Gly Ala TrpIle Phe Thr Gly Gly 210 215 220 Val Asn Thr Gly Val Ile Arg His Val GlyAsp Ala Leu Lys Asp His 225 230 235 240 Ala Ser Lys Ser Arg Gly Lys IleCys Thr Ile Gly Ile Ala Pro Trp 245 250 255 Gly Ile Val Glu Asn Gln GluAsp Leu Ile Gly Arg Asp Val Val Arg 260 265 270 Pro Tyr Gln Thr Met SerAsn Pro Met Ser Lys Leu Thr Val Leu Asn 275 280 285 Ser Met His Ser HisPhe Ile Leu Ala Asp Asn Gly Thr Thr Gly Lys 290 295 300 Tyr Gly Ala GluVal Lys Leu Arg Arg Gln Leu Glu Lys His Ile Ser 305 310 315 320 Leu GlnLys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro Val Val Ala 325 330 335 LeuIle Val Glu Gly Gly Pro Asn Val Ile Ser Ile Val Leu Glu Tyr 340 345 350Leu Arg Asp Thr Pro Pro Val Pro Val Val Val Cys Asp Gly Ser Gly 355 360365 Arg Ala Ser Asp Ile Leu Ala Phe Gly His Lys Tyr Ser Glu Glu Gly 370375 380 Gly Leu Ile Asn Glu Ser Leu Arg Asp Gln Leu Leu Val Thr Ile Gln385 390 395 400 Lys Thr Phe Thr Tyr Thr Arg Thr Gln Ala Gln His Leu PheIle Ile 405 410 415 Leu Met Glu Cys Met Lys Lys Lys Glu Leu Ile Thr ValPhe Arg Met 420 425 430 Gly Ser Glu Gly His Gln Asp Ile Asp Leu Ala IleLeu Thr Ala Leu 435 440 445 Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp GlnLeu Ser Leu Ala Leu 450 455 460 Ala Trp Asn Arg Val Asp Ile Ala Arg SerGln Ile Phe Ile Tyr Gly 465 470 475 480 Gln Gln Trp Pro Val Gly Ser LeuGlu Gln Ala Met Leu Asp Ala Leu 485 490 495 Val Leu Asp Arg Val Asp PheVal Lys Leu Leu Ile Glu Asn Gly Val 500 505 510 Ser Met His Arg Phe LeuThr Ile Ser Arg Leu Glu Glu Leu Tyr Asn 515 520 525 Thr Arg His Gly ProSer Asn Thr Leu Tyr His Leu Val Arg Asp Val 530 535 540 Lys Lys Arg GluTyr Pro Gly Phe Gly Trp Ile Tyr Phe Lys Gly Asn 545 550 555 560 Leu ProPro Asp Tyr Arg Ile Ser Leu Ile Asp Ile Gly Leu Val Ile 565 570 575 GluTyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr Thr Arg Lys Arg 580 585 590Phe Arg Thr Leu Tyr His Asn Leu Phe Gly Pro Lys Arg Asp Asp Ile 595 600605 Pro Leu Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg Glu Glu Glu Val 610615 620 Asp Ile Asp Leu Asp Asp Pro Glu Ile Asn His Phe Pro Phe Pro Phe625 630 635 640 His Glu Leu Met Val Trp Ala Val Leu Met Lys Arg Gln LysMet Ala 645 650 655 Leu Phe Phe Trp Gln His Gly Glu Glu Ala Met Ala LysAla Leu Val 660 665 670 Ala Cys Lys Leu Cys Lys Ala Met Ala His Glu AlaSer Glu Asn Asp 675 680 685 Met Val Asp Asp Ile Ser Gln Glu Leu Asn HisAsn Ser Arg Asp Phe 690 695 700 Gly Gln Leu Ala Val Glu Leu Leu Asp GlnSer Tyr Lys Gln Asp Glu 705 710 715 720 Gln Leu Ala Met Lys Leu Leu ThrTyr Glu Leu Lys Asn Trp Ser Asn 725 730 735 Ala Thr Cys Leu Gln Leu AlaVal Ala Ala Lys His Arg Asp Phe Ile 740 745 750 Ala His Thr Cys Ser GlnMet Leu Leu Thr Asp Met Trp Met Gly Arg 755 760 765 Leu Arg Met Arg LysAsn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu 770 775 780 Leu Pro Pro SerIle Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met 785 790 795 800 Pro TyrMet Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu Ala 805 810 815 GluGlu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp Met Glu 820 825 830Leu Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys 835 840845 Asp Glu Glu Glu Val Gln Ser Lys His Arg Leu Ile Pro Leu Gly Arg 850855 860 Lys Ile Tyr Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr865 870 875 880 Thr Leu Ala Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr IleVal Leu 885 890 895 Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu Trp IleVal Ile Ser 900 905 910 Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg GluIle Leu Met Ser 915 920 925 Glu Pro Gly Lys Leu Leu Gln Lys Val Lys ValTrp Leu Gln Glu Tyr 930 935 940 Trp Asn Val Thr Asp Leu Ile Ala Ile LeuLeu Phe Ser Val Gly Met 945 950 955 960 Ile Leu Arg Leu Gln Asp Gln ProPhe Arg Ser Asp Gly Arg Val Ile 965 970 975 Tyr Cys Val Asn Ile Ile TyrTrp Tyr Ile Arg Leu Leu Asp Ile Phe 980 985 990 Gly Val Asn Lys Tyr LeuGly Pro Tyr Val Met Met Ile Gly Lys Met 995 1000 1005 Met Ile Asp MetMet Tyr Phe Val Ile Ile Met Leu Val Val Leu 1010 1015 1020 Met Ser PheGly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu 1025 1030 1035 Glu ProSer Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr 1040 1045 1050 TrpMet Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro 1055 1060 1065Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu 1070 10751080 Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala 10851090 1095 Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile1100 1105 1110 Ala Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile SerAsn 1115 1120 1125 Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met ThrPhe His 1130 1135 1140 Glu Arg Pro Val Leu Pro Pro Pro Leu Ile Ile PheSer His Met 1145 1150 1155 Thr Met Ile Phe Gln His Leu Cys Cys Arg TrpArg Lys His Glu 1160 1165 1170 Ser Asp Pro Asp Glu Arg Asp Tyr Gly LeuLys Leu Phe Ile Thr 1175 1180 1185 Asp Asp Glu Leu Lys Lys Val His AspPhe Glu Glu Gln Cys Ile 1190 1195 1200 Glu Glu Tyr Phe Arg Glu Lys AspAsp Arg Phe Asn Ser Ser Asn 1205 1210 1215 Asp Glu Arg Ile Arg Val ThrSer Glu Arg Val Glu Asn Met Ser 1220 1225 1230 Met Arg Leu Glu Glu ValAsn Glu Arg Glu His Ser Met Lys Ala 1235 1240 1245 Ser Leu Gln Thr ValAsp Ile Arg Leu Ala Gln Leu Glu Asp Leu 1250 1255 1260 Ile Gly Arg MetAla Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu 1265 1270 1275 Arg Ala GluSer Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys 1280 1285 1290 Thr AspAla Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln 1295 1300 1305 GluGly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly 1310 1315 1320Glu Glu Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met 1325 13301335 Arg Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly 13401345 1350 Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu1355 1360 1365 His Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro LysAla 1370 1375 1380 Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro AspSer Arg 1385 1390 1395 Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser AlaMet Asp Glu 1400 1405 1410 Leu His Cys Asp Ile Asp Pro Leu Asp Asn SerVal Asn Ile Leu 1415 1420 1425 Gly Leu Gly Glu Pro Ser Phe Ser Thr ProVal Pro Ser Thr Ala 1430 1435 1440 Pro Ser Ser Ser Ala Tyr Ala Thr LeuAla Pro Thr Asp Arg Pro 1445 1450 1455 Pro Ser Arg Ser Ile Asp Phe GluAsp Ile Thr Ser Met Asp Thr 1460 1465 1470 Arg Ser Phe Ser Ser Asp TyrThr His Leu Pro Glu Cys Gln Asn 1475 1480 1485 Pro Trp Asp Ser Glu ProPro Met Tyr His Thr Ile Glu Arg Ser 1490 1495 1500 Lys Ser Ser Arg TyrLeu Ala Thr Thr Pro Phe Leu Leu Glu Glu 1505 1510 1515 Ala Pro Ile ValLys Ser His Ser Phe Met Phe Ser Pro Ser Arg 1520 1525 1530 Ser Tyr TyrAla Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr 1535 1540 1545 Thr SerIle Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro 1550 1555 1560 GlnAla Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp 1565 1570 1575Lys Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu 1580 15851590 Leu Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly 15951600 1605 Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser1610 1615 1620 Glu Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile GluArg 1625 1630 1635 Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro TyrAla His 1640 1645 1650 Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu AspArg Gln Arg 1655 1660 1665 Asn Thr Ala Ser Leu Gln Asn Pro Phe Gln ArgSer Lys Ser Ser 1670 1675 1680 Lys Pro Glu Gly Arg Gly Asp Ser Leu SerMet Arg Arg Leu Ser 1685 1690 1695 Arg Thr Ser Ala Phe Gln Ser Phe GluSer Lys His Thr 1700 1705 1710 11 5276 DNA Homo sapiens CDS (72)..(5273)11 cgcccgcggc gaggagccag cgagagcgct cggcgctggg ctgtttcccg gccgagggag 60gcgaacttct c atg ggg aag aag tgg agg gat gcg gcg gaa atg gag cgg 110 MetGly Lys Lys Trp Arg Asp Ala Ala Glu Met Glu Arg 1 5 10 ggc tgc tcc gaccgc gag gac aac gcg gag agc cgc aga cgc agc cgg 158 Gly Cys Ser Asp ArgGlu Asp Asn Ala Glu Ser Arg Arg Arg Ser Arg 15 20 25 agc gcc agc cgg ggcagg ttt gcc gag tcg tgg aaa agg tta agt tcc 206 Ser Ala Ser Arg Gly ArgPhe Ala Glu Ser Trp Lys Arg Leu Ser Ser 30 35 40 45 aag cag ggg tcc accaaa cgc tcg gga ctc ccg tcg cag cag acg ccg 254 Lys Gln Gly Ser Thr LysArg Ser Gly Leu Pro Ser Gln Gln Thr Pro 50 55 60 gct cag aaa tcc tgg atagaa aga gca ttt tat aaa aga gaa tgt gtc 302 Ala Gln Lys Ser Trp Ile GluArg Ala Phe Tyr Lys Arg Glu Cys Val 65 70 75 cac atc ata ccc agc acc aaagac ccc cat agg tgt tgc tgt ggg cgt 350 His Ile Ile Pro Ser Thr Lys AspPro His Arg Cys Cys Cys Gly Arg 80 85 90 ctg ata ggc cag cat gtt ggc ctcacc ccc agt atc tcc gtg ctt cag 398 Leu Ile Gly Gln His Val Gly Leu ThrPro Ser Ile Ser Val Leu Gln 95 100 105 aat gag aaa aat gaa agt cgc ctctcc cga aat gac atc cag tct gaa 446 Asn Glu Lys Asn Glu Ser Arg Leu SerArg Asn Asp Ile Gln Ser Glu 110 115 120 125 aag tgg tcc atc agc aaa cacact caa ctc agc cct acg gat gct ttt 494 Lys Trp Ser Ile Ser Lys His ThrGln Leu Ser Pro Thr Asp Ala Phe 130 135 140 ggg acc att gag ttc caa ggaggt ggc cat tcc aac aaa gcc atg tat 542 Gly Thr Ile Glu Phe Gln Gly GlyGly His Ser Asn Lys Ala Met Tyr 145 150 155 gtg cga gta tct ttt gat acaaaa cct gat ctc ctc tta cac ctg atg 590 Val Arg Val Ser Phe Asp Thr LysPro Asp Leu Leu Leu His Leu Met 160 165 170 acc aag gaa tgg cag ttg gagctt ccc aag ctt ctc atc tct gtc cat 638 Thr Lys Glu Trp Gln Leu Glu LeuPro Lys Leu Leu Ile Ser Val His 175 180 185 ggg ggc ctg cag aac ttt gaactc cag cca aaa ctc aag caa gtc ttt 686 Gly Gly Leu Gln Asn Phe Glu LeuGln Pro Lys Leu Lys Gln Val Phe 190 195 200 205 ggg aaa ggg ctc atc aaagca gca atg aca act gga gcg tgg ata ttc 734 Gly Lys Gly Leu Ile Lys AlaAla Met Thr Thr Gly Ala Trp Ile Phe 210 215 220 act gga ggg gtt aac acaggt gtt att cgt cat gtt ggc gat gcc ttg 782 Thr Gly Gly Val Asn Thr GlyVal Ile Arg His Val Gly Asp Ala Leu 225 230 235 aag gat cat gcc tct aagtct cga gga aag ata tgc acc ata ggt att 830 Lys Asp His Ala Ser Lys SerArg Gly Lys Ile Cys Thr Ile Gly Ile 240 245 250 gcc ccc tgg gga att gtggaa aac cag gag gac ctc att gga aga gat 878 Ala Pro Trp Gly Ile Val GluAsn Gln Glu Asp Leu Ile Gly Arg Asp 255 260 265 gtt gtc cgg cca tac cagacc atg tcc aat ccc atg agc aag ctc act 926 Val Val Arg Pro Tyr Gln ThrMet Ser Asn Pro Met Ser Lys Leu Thr 270 275 280 285 gtt ctc aac agc atgcat tcc cac ttc att ctg gct gac aac ggg acc 974 Val Leu Asn Ser Met HisSer His Phe Ile Leu Ala Asp Asn Gly Thr 290 295 300 act gga aaa tat ggagca gag gtg aaa ctt cga aga caa ctg gaa aag 1022 Thr Gly Lys Tyr Gly AlaGlu Val Lys Leu Arg Arg Gln Leu Glu Lys 305 310 315 cat att tca ctc cagaag ata aac aca aga tgc ctg ccg ttt ttc tct 1070 His Ile Ser Leu Gln LysIle Asn Thr Arg Cys Leu Pro Phe Phe Ser 320 325 330 ctt gac tcc cgc ttgttt tat tca ttt tgg ggt agt tgc cag cta gac 1118 Leu Asp Ser Arg Leu PheTyr Ser Phe Trp Gly Ser Cys Gln Leu Asp 335 340 345 tca gtt gga atc ggtcaa ggt gtt cct gtg gtg gca ctc ata gtg gaa 1166 Ser Val Gly Ile Gly GlnGly Val Pro Val Val Ala Leu Ile Val Glu 350 355 360 365 gga gga ccc aatgtg atc tcg att gtt ttg gag tac ctt cga gac acc 1214 Gly Gly Pro Asn ValIle Ser Ile Val Leu Glu Tyr Leu Arg Asp Thr 370 375 380 cct ccc gtg ccagtg gtt gtc tgt gat ggg agt gga cgg gca tcg gac 1262 Pro Pro Val Pro ValVal Val Cys Asp Gly Ser Gly Arg Ala Ser Asp 385 390 395 atc ctg gcc tttggg cat aaa tac tca gaa gaa ggc gga ctg ata aat 1310 Ile Leu Ala Phe GlyHis Lys Tyr Ser Glu Glu Gly Gly Leu Ile Asn 400 405 410 gaa tct ttg agggac cag ctg ttg gtg act ata cag aag act ttc aca 1358 Glu Ser Leu Arg AspGln Leu Leu Val Thr Ile Gln Lys Thr Phe Thr 415 420 425 tac act cga acccaa gct cag cat ctg ttc atc atc ctc atg gag tgc 1406 Tyr Thr Arg Thr GlnAla Gln His Leu Phe Ile Ile Leu Met Glu Cys 430 435 440 445 atg aag aagaag gaa ttg att acg gta ttt cgg atg gga tca gaa gga 1454 Met Lys Lys LysGlu Leu Ile Thr Val Phe Arg Met Gly Ser Glu Gly 450 455 460 cac cag gacatt gat ttg gct atc ctg aca gct tta ctc aaa gga gcc 1502 His Gln Asp IleAsp Leu Ala Ile Leu Thr Ala Leu Leu Lys Gly Ala 465 470 475 aat gcc tcggcc cca gac caa ctg agc tta gct tta gcc tgg aac aga 1550 Asn Ala Ser AlaPro Asp Gln Leu Ser Leu Ala Leu Ala Trp Asn Arg 480 485 490 gtc gac atcgct cgc agc cag atc ttt att tac ggg caa cag tgg ccg 1598 Val Asp Ile AlaArg Ser Gln Ile Phe Ile Tyr Gly Gln Gln Trp Pro 495 500 505 gtg gga tctctg gag caa gcc atg ttg gat gcc tta gtt ctg gac aga 1646 Val Gly Ser LeuGlu Gln Ala Met Leu Asp Ala Leu Val Leu Asp Arg 510 515 520 525 gtg gatttt gtg aaa tta ctc ata gag aat gga gta agc atg cac cgt 1694 Val Asp PheVal Lys Leu Leu Ile Glu Asn Gly Val Ser Met His Arg 530 535 540 ttt ctcacc atc tcc aga cta gag gaa ttg tac aat acg aga cat ggg 1742 Phe Leu ThrIle Ser Arg Leu Glu Glu Leu Tyr Asn Thr Arg His Gly 545 550 555 ccc tcaaat aca ttg tac cac ttg gtc agg gat gtc aaa aag ggg aac 1790 Pro Ser AsnThr Leu Tyr His Leu Val Arg Asp Val Lys Lys Gly Asn 560 565 570 ctg ccccca gac tac aga atc agc ctg att gac atc ggc ctg gtg atc 1838 Leu Pro ProAsp Tyr Arg Ile Ser Leu Ile Asp Ile Gly Leu Val Ile 575 580 585 gag tacctg atg ggc ggg gct tat cgc tgc aac tac acg cgc aag cgc 1886 Glu Tyr LeuMet Gly Gly Ala Tyr Arg Cys Asn Tyr Thr Arg Lys Arg 590 595 600 605 ttccgg acc ctc tac cac aac ctc ttc ggc ccc aag agg ccc aaa gcc 1934 Phe ArgThr Leu Tyr His Asn Leu Phe Gly Pro Lys Arg Pro Lys Ala 610 615 620 ttgaaa ctg ctg gga atg gag gat gat att ccc ttg agg cga gga aga 1982 Leu LysLeu Leu Gly Met Glu Asp Asp Ile Pro Leu Arg Arg Gly Arg 625 630 635 aagaca acc aag aaa cgt gaa gaa gag gtg gac att gac ttg gat gat 2030 Lys ThrThr Lys Lys Arg Glu Glu Glu Val Asp Ile Asp Leu Asp Asp 640 645 650 cctgag atc aac cac ttc ccc ttc cct ttc cat gag ctc atg gtg tgg 2078 Pro GluIle Asn His Phe Pro Phe Pro Phe His Glu Leu Met Val Trp 655 660 665 gctgtt ctc atg aag cgg cag aag atg gcc ctg ttc ttc tgg cag cac 2126 Ala ValLeu Met Lys Arg Gln Lys Met Ala Leu Phe Phe Trp Gln His 670 675 680 685ggt gag gag gcc atg gcc aag gcc ctg gtg gcc tgc aag ctc tgc aaa 2174 GlyGlu Glu Ala Met Ala Lys Ala Leu Val Ala Cys Lys Leu Cys Lys 690 695 700gcc atg gct cat gag gcc tct gag aac gac atg gtt gac gac att tcc 2222 AlaMet Ala His Glu Ala Ser Glu Asn Asp Met Val Asp Asp Ile Ser 705 710 715cag gag ctg aat cac aat tcc aga gac ttt ggc cag ctg gct gtg gag 2270 GlnGlu Leu Asn His Asn Ser Arg Asp Phe Gly Gln Leu Ala Val Glu 720 725 730ctc ctg gac cag tcc tac aag cag gac gaa cag ctg gcc atg aaa ctg 2318 LeuLeu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu Ala Met Lys Leu 735 740 745ctg acg tat gag ctg aag aac tgg agc aac gcc acg tgc ctg cag ctt 2366 LeuThr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys Leu Gln Leu 750 755 760765 gcc gtg gct gcc aaa cac cgc gac ttc atc gcg cac acg tgc agc cag 2414Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr Cys Ser Gln 770 775780 atg ctg ctc acc gac atg tgg atg ggc cgg ctc cgc atg cgc aag aac 2462Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn 785 790795 tca ggc ctc aag gta att ctg gga att cta ctt cct cct tca att ctc 2510Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu 800 805810 agc ttg gag ttc aag aac aaa gac gac atg ccc tat atg tct cag gcc 2558Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala 815 820825 cag gaa atc cac ctc caa gag aag gag gca gaa gaa cca gag aag ccc 2606Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro 830 835840 845 aca aag gaa aaa gag gaa gag gac atg gag ctc aca gca atg ttg gga2654 Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly 850855 860 cga aac aac ggg gag tcc tcc agg aag aag gat gaa gag gaa gtt cag2702 Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln 865870 875 agc aag cac cgg tta atc ccc ctc ggc aga aaa atc tat gaa ttc tac2750 Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr 880885 890 aat gca ccc atc gtg aag ttc tgg ttc tac aca ctg gcg tat atc gga2798 Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly 895900 905 tac ctg atg ctc ttc aac tat atc gtg tta gtg aag atg gaa cgc tgg2846 Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp 910915 920 925 ccg tcc acc cag gaa tgg atc gta atc tcc tat att ttc acc ctggga 2894 Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly930 935 940 ata gaa aag atg aga gag att ctg atg tca gag cca ggg aag ttgcta 2942 Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu945 950 955 cag aaa gtg aag gta tgg ctg cag gag tac tgg aat gtc acg gacctc 2990 Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp Leu960 965 970 atc gcc atc ctt ctg ttt tct gtc gga atg atc ctt cgt ctc caagac 3038 Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln Asp975 980 985 cag ccc ttc agg agt gac ggg agg gtc atc tac tgc gtg aac atcatt 3086 Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile Ile990 995 1000 1005 tac tgg tat atc cgt ctc cta gac atc ttc ggc gtg aacaag tat 3131 Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys Tyr1010 1015 1020 ttg ggc ccg tat gta atg atg att gga aaa atg atg ata gacatg 3176 Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met1025 1030 1035 atg tac ttt gtc atc att atg ctg gtg gtt ctg atg agc tttggg 3221 Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser Phe Gly1040 1045 1050 gtc gcc agg caa gcc atc ctt ttt ccc aat gag gag cca tcatgg 3266 Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp1055 1060 1065 aaa ctg gcc aag aac atc ttc tac atg ccc tat tgg atg atttat 3311 Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr1070 1075 1080 ggg gaa gtg ttt gcg gac cag ata gac cct ccc tgt gga cagaat 3356 Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn1085 1090 1095 gag acc cga gag gat ggt aaa ata atc cag ctg cct ccc tgcaag 3401 Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys1100 1105 1110 aca gga gct tgg atc gtg ccg gcc atc atg gcc tgc tac ctctta 3446 Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys Tyr Leu Leu1115 1120 1125 gtg gca aac atc ttg ctg gtc aac ctc ctc att gct gtc tttaac 3491 Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Val Phe Asn1130 1135 1140 aat aca ttt ttt gaa gta aaa tcg ata tcc aac caa gtc tggaag 3536 Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp Lys1145 1150 1155 ttt cag agg tat cag ctc atc atg act ttc cat gaa agg ccagtt 3581 Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg Pro Val1160 1165 1170 ctg ccc cca cca ctg atc atc ttc agc cac atg acc atg atattc 3626 Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met Ile Phe1175 1180 1185 cag cac ctg tgc tgc cga tgg agg aaa cac gag agc gac ccggat 3671 Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser Asp Pro Asp1190 1195 1200 gaa agg gac tac ggc ctg aaa ctc ttc ata acc gat gat gagctc 3716 Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp Glu Leu1205 1210 1215 aag aaa gta cat gac ttt gaa gag caa tgc ata gaa gaa tacttc 3761 Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe1220 1225 1230 aga gaa aag gat gat cgg ttc aac tca tct aat gat gag aggata 3806 Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile1235 1240 1245 cgg gtg act tca gaa agg gtg gag aac atg tct atg cgg ctggag 3851 Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu1250 1255 1260 gaa gtc aac gag aga gag cac tcc atg aag gct tca ctc cagacc 3896 Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr1265 1270 1275 gtg gac atc cgg ctg gcg cag ctg gaa gac ctt atc ggg cgcatg 3941 Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met1280 1285 1290 gcc acg gcc ctg gag cgc ctg aca ggt ctg gag cgg gcc gagtcc 3986 Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser1295 1300 1305 aac aaa atc cgc tcg agg acc tcg tca gac tgc acg gac gccgcc 4031 Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala1310 1315 1320 tac att gtc cgt cag agc agc ttc aac agc cag gaa ggg aacacc 4076 Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr1325 1330 1335 ttc aag ctc caa gag agt ata gac cct gca ggt gag gag accatg 4121 Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met1340 1345 1350 tcc cca act tct cca acc tta atg ccc cgt atg cga agc cattct 4166 Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg Ser His Ser1355 1360 1365 ttc tat tca gtc aat atg aaa gac aaa ggt ggt ata gaa aagttg 4211 Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile Glu Lys Leu1370 1375 1380 gaa agt att ttt aaa gaa agg tcc ctg agc cta cac cgg gctact 4256 Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His Arg Ala Thr1385 1390 1395 agt tcc cac tct gta gca aaa gaa ccc aaa gct cct gca gcccct 4301 Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro1400 1405 1410 gcc aac acc ttg gcc att gtt cct gat tcc aga aga cca tcatcg 4346 Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser Ser1415 1420 1425 tgt ata gac atc tat gtc tct gct atg gat gag ctc cac tgtgat 4391 Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His Cys Asp1430 1435 1440 ata gac cct ctg gac aat tcc gtg aac atc ctt ggg cta ggcgag 4436 Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu Gly Glu1445 1450 1455 cca agc ttt tca act cca gta cct tcc aca gcc cct tca agtagt 4481 Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser Ser Ser1460 1465 1470 gcc tat gca aca ctt gca ccc aca gac aga cct cca agc cggagc 4526 Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser1475 1480 1485 att gat ttt gag gac atc acc tcc atg gac act aga tct ttttct 4571 Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser1490 1495 1500 tca gac tac acc cac ctc cca gaa tgc caa aac ccc tgg gactca 4616 Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser1505 1510 1515 gag cct ccg atg tac cac acc att gag cgt tcc aaa agt agccgc 4661 Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg1520 1525 1530 tac cta gcc acc aca ccc ttt ctt cta gaa gag gct ccc attgtg 4706 Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val1535 1540 1545 aaa tct cat agc ttt atg ttt tcc ccc tca agg agc tat tatgcc 4751 Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala1550 1555 1560 aac ttt ggg gtg cct gta aaa aca gca gaa tac aca agt attaca 4796 Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr1565 1570 1575 gac tgt att gac aca agg tgt gtc aat gcc cct caa gca attgcg 4841 Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala Ile Ala1580 1585 1590 gac aga gct gcc ttc cct gga ggt ctt gga gac aaa gtg gaggac 4886 Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp1595 1600 1605 tta act tgc tgc cat cca gag cga gaa gca gaa ctg agt cacccc 4931 Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu Ser His Pro1610 1615 1620 agc tct gac agt gag gag aat gag gcc aaa ggc cgc aga gccacc 4976 Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr1625 1630 1635 att gca ata tcc tcc cag gag ggt gat aac tca gag aga accctg 5021 Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu1640 1645 1650 tcc aac aac atc act gtt ccc aag ata gag cgc gcc aac agctac 5066 Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr1655 1660 1665 tcg gca gag gag cca agt gcg cca tat gca cac acc agg aagagc 5111 Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr Arg Lys Ser1670 1675 1680 ttc tcc atc agt gac aaa ctc gac agg cag cgg aac aca gcaagc 5156 Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr Ala Ser1685 1690 1695 ctg caa aat ccc ttc cag aga agc aag tcc tcc aag ccg gagggc 5201 Leu Gln Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro Glu Gly1700 1705 1710 cga ggg gac agc ctg tcc atg agg aga ctg tcc aga aca tcggct 5246 Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg Thr Ser Ala1715 1720 1725 ttc caa agc ttt gaa agc aag cac acc taa 5276 Phe Gln SerPhe Glu Ser Lys His Thr 1730 12 1734 PRT Homo sapiens 12 Met Gly Lys LysTrp Arg Asp Ala Ala Glu Met Glu Arg Gly Cys Ser 1 5 10 15 Asp Arg GluAsp Asn Ala Glu Ser Arg Arg Arg Ser Arg Ser Ala Ser 20 25 30 Arg Gly ArgPhe Ala Glu Ser Trp Lys Arg Leu Ser Ser Lys Gln Gly 35 40 45 Ser Thr LysArg Ser Gly Leu Pro Ser Gln Gln Thr Pro Ala Gln Lys 50 55 60 Ser Trp IleGlu Arg Ala Phe Tyr Lys Arg Glu Cys Val His Ile Ile 65 70 75 80 Pro SerThr Lys Asp Pro His Arg Cys Cys Cys Gly Arg Leu Ile Gly 85 90 95 Gln HisVal Gly Leu Thr Pro Ser Ile Ser Val Leu Gln Asn Glu Lys 100 105 110 AsnGlu Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu Lys Trp Ser 115 120 125Ile Ser Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe Gly Thr Ile 130 135140 Glu Phe Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr Val Arg Val 145150 155 160 Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met Thr LysGlu 165 170 175 Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His GlyGly Leu 180 185 190 Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln Val PheGly Lys Gly 195 200 205 Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp IlePhe Thr Gly Gly 210 215 220 Val Asn Thr Gly Val Ile Arg His Val Gly AspAla Leu Lys Asp His 225 230 235 240 Ala Ser Lys Ser Arg Gly Lys Ile CysThr Ile Gly Ile Ala Pro Trp 245 250 255 Gly Ile Val Glu Asn Gln Glu AspLeu Ile Gly Arg Asp Val Val Arg 260 265 270 Pro Tyr Gln Thr Met Ser AsnPro Met Ser Lys Leu Thr Val Leu Asn 275 280 285 Ser Met His Ser His PheIle Leu Ala Asp Asn Gly Thr Thr Gly Lys 290 295 300 Tyr Gly Ala Glu ValLys Leu Arg Arg Gln Leu Glu Lys His Ile Ser 305 310 315 320 Leu Gln LysIle Asn Thr Arg Cys Leu Pro Phe Phe Ser Leu Asp Ser 325 330 335 Arg LeuPhe Tyr Ser Phe Trp Gly Ser Cys Gln Leu Asp Ser Val Gly 340 345 350 IleGly Gln Gly Val Pro Val Val Ala Leu Ile Val Glu Gly Gly Pro 355 360 365Asn Val Ile Ser Ile Val Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val 370 375380 Pro Val Val Val Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala 385390 395 400 Phe Gly His Lys Tyr Ser Glu Glu Gly Gly Leu Ile Asn Glu SerLeu 405 410 415 Arg Asp Gln Leu Leu Val Thr Ile Gln Lys Thr Phe Thr TyrThr Arg 420 425 430 Thr Gln Ala Gln His Leu Phe Ile Ile Leu Met Glu CysMet Lys Lys 435 440 445 Lys Glu Leu Ile Thr Val Phe Arg Met Gly Ser GluGly His Gln Asp 450 455 460 Ile Asp Leu Ala Ile Leu Thr Ala Leu Leu LysGly Ala Asn Ala Ser 465 470 475 480 Ala Pro Asp Gln Leu Ser Leu Ala LeuAla Trp Asn Arg Val Asp Ile 485 490 495 Ala Arg Ser Gln Ile Phe Ile TyrGly Gln Gln Trp Pro Val Gly Ser 500 505 510 Leu Glu Gln Ala Met Leu AspAla Leu Val Leu Asp Arg Val Asp Phe 515 520 525 Val Lys Leu Leu Ile GluAsn Gly Val Ser Met His Arg Phe Leu Thr 530 535 540 Ile Ser Arg Leu GluGlu Leu Tyr Asn Thr Arg His Gly Pro Ser Asn 545 550 555 560 Thr Leu TyrHis Leu Val Arg Asp Val Lys Lys Gly Asn Leu Pro Pro 565 570 575 Asp TyrArg Ile Ser Leu Ile Asp Ile Gly Leu Val Ile Glu Tyr Leu 580 585 590 MetGly Gly Ala Tyr Arg Cys Asn Tyr Thr Arg Lys Arg Phe Arg Thr 595 600 605Leu Tyr His Asn Leu Phe Gly Pro Lys Arg Pro Lys Ala Leu Lys Leu 610 615620 Leu Gly Met Glu Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys Thr Thr 625630 635 640 Lys Lys Arg Glu Glu Glu Val Asp Ile Asp Leu Asp Asp Pro GluIle 645 650 655 Asn His Phe Pro Phe Pro Phe His Glu Leu Met Val Trp AlaVal Leu 660 665 670 Met Lys Arg Gln Lys Met Ala Leu Phe Phe Trp Gln HisGly Glu Glu 675 680 685 Ala Met Ala Lys Ala Leu Val Ala Cys Lys Leu CysLys Ala Met Ala 690 695 700 His Glu Ala Ser Glu Asn Asp Met Val Asp AspIle Ser Gln Glu Leu 705 710 715 720 Asn His Asn Ser Arg Asp Phe Gly GlnLeu Ala Val Glu Leu Leu Asp 725 730 735 Gln Ser Tyr Lys Gln Asp Glu GlnLeu Ala Met Lys Leu Leu Thr Tyr 740 745 750 Glu Leu Lys Asn Trp Ser AsnAla Thr Cys Leu Gln Leu Ala Val Ala 755 760 765 Ala Lys His Arg Asp PheIle Ala His Thr Cys Ser Gln Met Leu Leu 770 775 780 Thr Asp Met Trp MetGly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu 785 790 795 800 Lys Val IleLeu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu 805 810 815 Phe LysAsn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile 820 825 830 HisLeu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu 835 840 845Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn Asn 850 855860 Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His 865870 875 880 Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn AlaPro 885 890 895 Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly TyrLeu Met 900 905 910 Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg TrpPro Ser Thr 915 920 925 Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr LeuGly Ile Glu Lys 930 935 940 Met Arg Glu Ile Leu Met Ser Glu Pro Gly LysLeu Leu Gln Lys Val 945 950 955 960 Lys Val Trp Leu Gln Glu Tyr Trp AsnVal Thr Asp Leu Ile Ala Ile 965 970 975 Leu Leu Phe Ser Val Gly Met IleLeu Arg Leu Gln Asp Gln Pro Phe 980 985 990 Arg Ser Asp Gly Arg Val IleTyr Cys Val Asn Ile Ile Tyr Trp Tyr 995 1000 1005 Ile Arg Leu Leu AspIle Phe Gly Val Asn Lys Tyr Leu Gly Pro 1010 1015 1020 Tyr Val Met MetIle Gly Lys Met Met Ile Asp Met Met Tyr Phe 1025 1030 1035 Val Ile IleMet Leu Val Val Leu Met Ser Phe Gly Val Ala Arg 1040 1045 1050 Gln AlaIle Leu Phe Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala 1055 1060 1065 LysAsn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val 1070 1075 1080Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg 1085 10901095 Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala 11001105 1110 Trp Ile Val Pro Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn1115 1120 1125 Ile Leu Leu Val Asn Leu Leu Ile Ala Val Phe Asn Asn ThrPhe 1130 1135 1140 Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp Lys PheGln Arg 1145 1150 1155 Tyr Gln Leu Ile Met Thr Phe His Glu Arg Pro ValLeu Pro Pro 1160 1165 1170 Pro Leu Ile Ile Phe Ser His Met Thr Met IlePhe Gln His Leu 1175 1180 1185 Cys Cys Arg Trp Arg Lys His Glu Ser AspPro Asp Glu Arg Asp 1190 1195 1200 Tyr Gly Leu Lys Leu Phe Ile Thr AspAsp Glu Leu Lys Lys Val 1205 1210 1215 His Asp Phe Glu Glu Gln Cys IleGlu Glu Tyr Phe Arg Glu Lys 1220 1225 1230 Asp Asp Arg Phe Asn Ser SerAsn Asp Glu Arg Ile Arg Val Thr 1235 1240 1245 Ser Glu Arg Val Glu AsnMet Ser Met Arg Leu Glu Glu Val Asn 1250 1255 1260 Glu Arg Glu His SerMet Lys Ala Ser Leu Gln Thr Val Asp Ile 1265 1270 1275 Arg Leu Ala GlnLeu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala 1280 1285 1290 Leu Glu ArgLeu Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile 1295 1300 1305 Arg SerArg Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val 1310 1315 1320 ArgGln Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu 1325 1330 1335Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr 1340 13451350 Ser Pro Thr Leu Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser 13551360 1365 Val Asn Met Lys Asp Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile1370 1375 1380 Phe Lys Glu Arg Ser Leu Ser Leu His Arg Ala Thr Ser SerHis 1385 1390 1395 Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro AlaAsn Thr 1400 1405 1410 Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser SerCys Ile Asp 1415 1420 1425 Ile Tyr Val Ser Ala Met Asp Glu Leu His CysAsp Ile Asp Pro 1430 1435 1440 Leu Asp Asn Ser Val Asn Ile Leu Gly LeuGly Glu Pro Ser Phe 1445 1450 1455 Ser Thr Pro Val Pro Ser Thr Ala ProSer Ser Ser Ala Tyr Ala 1460 1465 1470 Thr Leu Ala Pro Thr Asp Arg ProPro Ser Arg Ser Ile Asp Phe 1475 1480 1485 Glu Asp Ile Thr Ser Met AspThr Arg Ser Phe Ser Ser Asp Tyr 1490 1495 1500 Thr His Leu Pro Glu CysGln Asn Pro Trp Asp Ser Glu Pro Pro 1505 1510 1515 Met Tyr His Thr IleGlu Arg Ser Lys Ser Ser Arg Tyr Leu Ala 1520 1525 1530 Thr Thr Pro PheLeu Leu Glu Glu Ala Pro Ile Val Lys Ser His 1535 1540 1545 Ser Phe MetPhe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly 1550 1555 1560 Val ProVal Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile 1565 1570 1575 AspThr Arg Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala 1580 1585 1590Ala Phe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys 1595 16001605 Cys His Pro Glu Arg Glu Ala Glu Leu Ser His Pro Ser Ser Asp 16101615 1620 Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile1625 1630 1635 Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu Ser AsnAsn 1640 1645 1650 Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr SerAla Glu 1655 1660 1665 Glu Pro Ser Ala Pro Tyr Ala His Thr Arg Lys SerPhe Ser Ile 1670 1675 1680 Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr AlaSer Leu Gln Asn 1685 1690 1695 Pro Phe Gln Arg Ser Lys Ser Ser Lys ProGlu Gly Arg Gly Asp 1700 1705 1710 Ser Leu Ser Met Arg Arg Leu Ser ArgThr Ser Ala Phe Gln Ser 1715 1720 1725 Phe Glu Ser Lys His Thr 1730 131533 PRT Homo sapiens 13 Met Tyr Ile Arg Val Ser Tyr Asp Thr Lys Pro AspSer Leu Leu His 1 5 10 15 Leu Met Val Lys Asp Trp Gln Leu Glu Leu ProLys Leu Leu Ile Ser 20 25 30 Val His Gly Gly Leu Gln Asn Phe Glu Met GlnPro Lys Leu Lys Gln 35 40 45 Val Phe Gly Lys Gly Leu Ile Lys Ala Ala MetThr Thr Gly Ala Trp 50 55 60 Ile Phe Thr Gly Gly Val Ser Thr Gly Val IleSer His Val Gly Asp 65 70 75 80 Ala Leu Lys Asp His Ser Ser Lys Ser ArgGly Arg Val Cys Ala Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile Val Glu AsnLys Glu Asp Leu Val Gly 100 105 110 Lys Asp Val Thr Arg Val Tyr Gln ThrMet Ser Asn Pro Leu Ser Lys 115 120 125 Leu Ser Val Leu Asn Asn Ser HisThr His Phe Ile Leu Ala Asp Asn 130 135 140 Gly Thr Leu Gly Lys Tyr GlyAla Glu Val Lys Leu Arg Arg Leu Leu 145 150 155 160 Glu Lys His Ile SerLeu Gln Lys Ile Asn Thr Arg Leu Gly Gln Gly 165 170 175 Val Pro Leu ValGly Leu Val Val Glu Gly Gly Pro Asn Val Val Ser 180 185 190 Ile Val LeuGlu Tyr Leu Gln Glu Glu Pro Pro Ile Pro Val Val Ile 195 200 205 Cys AspGly Ser Gly Arg Ala Ser Asp Ile Leu Ser Phe Ala His Lys 210 215 220 TyrCys Glu Glu Gly Gly Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu 225 230 235240 Leu Val Thr Ile Gln Lys Thr Phe Asn Tyr Asn Lys Ala Gln Ser His 245250 255 Gln Leu Phe Ala Ile Ile Met Glu Cys Met Lys Lys Lys Glu Leu Val260 265 270 Thr Val Phe Arg Met Gly Ser Glu Gly Gln Gln Asp Ile Glu MetAla 275 280 285 Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Val Ser Ala ProAsp Gln 290 295 300 Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile AlaArg Ser Gln 305 310 315 320 Ile Phe Val Phe Gly Pro His Trp Thr Pro LeuGly Ser Leu Ala Pro 325 330 335 Pro Thr Asp Ser Lys Ala Thr Glu Lys GluLys Lys Pro Pro Met Ala 340 345 350 Thr Thr Lys Gly Gly Arg Gly Lys GlyLys Gly Lys Lys Lys Gly Lys 355 360 365 Val Lys Glu Glu Val Glu Glu GluThr Asp Pro Arg Lys Ile Glu Leu 370 375 380 Leu Asn Trp Val Asn Ala LeuGlu Gln Ala Met Leu Asp Ala Leu Val 385 390 395 400 Leu Asp Arg Val AspPhe Val Lys Leu Leu Ile Glu Asn Gly Val Asn 405 410 415 Met Gln His PheLeu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr 420 425 430 Arg Leu GlyPro Pro Asn Thr Leu His Leu Leu Val Arg Asp Val Lys 435 440 445 Lys SerAsn Leu Pro Pro Asp Tyr His Ile Ser Leu Ile Asp Ile Gly 450 455 460 LeuVal Leu Glu Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr Thr 465 470 475480 Arg Lys Asn Phe Arg Thr Leu Tyr Asn Asn Leu Phe Gly Pro Lys Arg 485490 495 Pro Lys Ala Leu Lys Leu Leu Gly Met Glu Asp Asp Glu Pro Pro Ala500 505 510 Lys Gly Lys Lys Lys Lys Lys Lys Lys Lys Glu Glu Glu Ile AspIle 515 520 525 Asp Val Asp Asp Pro Ala Val Ser Arg Phe Gln Tyr Pro PheHis Glu 530 535 540 Leu Met Val Trp Ala Val Leu Met Lys Arg Gln Lys MetAla Val Phe 545 550 555 560 Leu Trp Gln Arg Gly Glu Glu Ser Met Ala LysAla Leu Val Ala Cys 565 570 575 Lys Leu Tyr Lys Ala Met Ala His Glu SerSer Glu Ser Asp Leu Val 580 585 590 Asp Asp Ile Ser Gln Asp Leu Asp AsnAsn Ser Lys Asp Phe Gly Gln 595 600 605 Leu Ala Leu Glu Leu Leu Asp GlnSer Tyr Lys His Asp Glu Gln Ile 610 615 620 Ala Met Lys Leu Leu Thr TyrGlu Leu Lys Asn Trp Ser Asn Ser Thr 625 630 635 640 Cys Leu Lys Leu AlaVal Ala Ala Lys His Arg Asp Phe Ile Ala His 645 650 655 Thr Cys Ser GlnMet Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg 660 665 670 Met Arg LysAsn Pro Gly Leu Lys Val Ile Met Gly Ile Leu Leu Pro 675 680 685 Pro ThrIle Leu Phe Leu Glu Phe Arg Thr Tyr Asp Asp Phe Ser Tyr 690 695 700 GlnThr Ser Lys Glu Asn Glu Asp Gly Lys Glu Lys Glu Glu Glu Asn 705 710 715720 Thr Asp Ala Asn Ala Asp Ala Gly Ser Arg Lys Gly Asp Glu Glu Asn 725730 735 Glu His Lys Lys Gln Arg Ser Ile Pro Ile Gly Thr Lys Ile Cys Glu740 745 750 Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Ile SerTyr 755 760 765 Leu Gly Tyr Leu Leu Leu Phe Asn Tyr Val Ile Leu Val ArgMet Asp 770 775 780 Gly Trp Pro Ser Leu Gln Glu Trp Ile Val Ile Ser TyrIle Val Ser 785 790 795 800 Leu Ala Leu Glu Lys Ile Arg Glu Ile Leu MetSer Glu Pro Gly Lys 805 810 815 Leu Ser Gln Lys Ile Lys Val Trp Leu GlnGlu Tyr Trp Asn Ile Thr 820 825 830 Asp Leu Val Ala Ile Ser Thr Phe MetIle Gly Ala Ile Leu Arg Leu 835 840 845 Gln Asn Gln Pro Tyr Met Gly TyrGly Arg Val Ile Tyr Cys Val Asp 850 855 860 Ile Ile Phe Trp Tyr Ile ArgVal Leu Asp Ile Phe Gly Val Asn Lys 865 870 875 880 Tyr Leu Gly Pro TyrVal Met Met Ile Gly Lys Met Met Ile Asp Met 885 890 895 Leu Tyr Phe ValVal Ile Met Leu Val Val Leu Met Ser Phe Gly Val 900 905 910 Ala Arg GlnAla Ile Leu His Pro Glu Glu Lys Pro Ser Trp Lys Leu 915 920 925 Ala ArgAsn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val 930 935 940 PheAla Asp Gln Ile Asp Leu Tyr Ala Met Glu Ile Asn Pro Pro Cys 945 950 955960 Gly Glu Asn Leu Tyr Asp Glu Glu Gly Lys Arg Leu Pro Pro Cys Ile 965970 975 Pro Gly Ala Trp Leu Thr Pro Ala Leu Met Ala Cys Tyr Leu Leu Val980 985 990 Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Val Phe Asn AsnThr 995 1000 1005 Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp LysPhe Gln 1010 1015 1020 Arg Tyr Gln Leu Ile Met Thr Phe His Asp Arg ProVal Leu Pro 1025 1030 1035 Pro Pro Met Ile Ile Leu Ser His Ile Tyr IleIle Ile Met Arg 1040 1045 1050 Leu Ser Gly Arg Cys Arg Lys Lys Arg GluGly Asp Gln Glu Glu 1055 1060 1065 Arg Asp Arg Gly Leu Lys Leu Phe LeuSer Asp Glu Glu Leu Lys 1070 1075 1080 Arg Leu His Glu Phe Glu Glu GlnCys Val Gln Glu His Phe Arg 1085 1090 1095 Glu Lys Glu Asp Glu Gln GlnSer Ser Ser Asp Glu Arg Ile Arg 1100 1105 1110 Val Thr Ser Glu Arg ValGlu Asn Met Ser Met Arg Leu Glu Glu 1115 1120 1125 Ile Asn Glu Arg GluThr Phe Met Lys Thr Ser Leu Gln Thr Val 1130 1135 1140 Asp Leu Arg LeuAla Gln Leu Glu Glu Leu Ser Asn Arg Met Val 1145 1150 1155 Asn Ala LeuGlu Asn Leu Ala Gly Ile Asp Arg Ser Asp Leu Ile 1160 1165 1170 Gln AlaArg Ser Arg Ala Ser Ser Glu Cys Glu Ala Thr Tyr Leu 1175 1180 1185 LeuArg Gln Ser Ser Ile Asn Ser Ala Asp Gly Tyr Ser Leu Tyr 1190 1195 1200Arg Tyr His Phe Asn Gly Glu Glu Leu Leu Phe Glu Asp Thr Ser 1205 12101215 Leu Ser Thr Ser Pro Gly Thr Gly Val Arg Lys Lys Thr Cys Ser 12201225 1230 Phe Arg Ile Lys Glu Glu Lys Asp Val Lys Thr His Leu Val Pro1235 1240 1245 Glu Cys Gln Asn Ser Leu His Leu Ser Leu Gly Thr Ser ThrSer 1250 1255 1260 Ala Thr Pro Asp Gly Ser His Leu Ala Val Asp Asp LeuLys Asn 1265 1270 1275 Ala Glu Glu Ser Lys Leu Gly Pro Asp Ile Gly IleSer Lys Glu 1280 1285 1290 Asp Asp Glu Arg Gln Thr Asp Ser Lys Lys GluGlu Thr Ile Ser 1295 1300 1305 Pro Ser Leu Asn Lys Thr Asp Val Ile HisGly Gln Asp Lys Ser 1310 1315 1320 Asp Val Gln Asn Thr Gln Leu Thr ValGlu Thr Thr Asn Ile Glu 1325 1330 1335 Gly Thr Ile Ser Tyr Pro Leu GluGlu Thr Lys Ile Thr Arg Tyr 1340 1345 1350 Phe Pro Asp Glu Thr Ile AsnAla Cys Lys Thr Met Lys Ser Arg 1355 1360 1365 Ser Phe Val Tyr Ser ArgGly Arg Lys Leu Val Gly Gly Val Asn 1370 1375 1380 Gln Asp Val Glu TyrSer Ser Ile Thr Asp Gln Gln Leu Thr Thr 1385 1390 1395 Glu Trp Gln CysGln Val Gln Lys Ile Thr Arg Ser His Ser Thr 1400 1405 1410 Asp Ile ProTyr Ile Val Ser Glu Ala Ala Val Gln Ala Glu Gln 1415 1420 1425 Lys GluGln Phe Ala Asp Met Gln Asp Glu His His Val Ala Glu 1430 1435 1440 AlaIle Pro Arg Ile Pro Arg Leu Ser Leu Thr Ile Thr Asp Arg 1445 1450 1455Asn Gly Met Glu Asn Leu Leu Ser Val Lys Pro Asp Gln Thr Leu 1460 14651470 Gly Phe Pro Ser Leu Arg Ser Lys Ser Leu His Gly His Pro Arg 14751480 1485 Asn Val Lys Ser Ile Gln Gly Lys Leu Asp Arg Ser Gly His Ala1490 1495 1500 Ser Ser Val Ser Ser Leu Val Ile Val Ser Gly Met Thr AlaGlu 1505 1510 1515 Glu Lys Lys Val Lys Lys Glu Lys Ala Ser Thr Glu ThrGlu Cys 1520 1525 1530 14 4665 DNA Homo sapiens CDS (1)..(4662) 14 atgtat gtg cga gta tct ttt gat aca aaa cct gat ctc ctc tta cac 48 Met TyrVal Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His 1 5 10 15 ctgatg acc aag gaa tgg cag ttg gag ctt ccc aag ctt ctc atc tct 96 Leu MetThr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 gtc catggg ggc ctg cag aac ttt gaa ctc cag cca aaa ctc aag caa 144 Val His GlyGly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln 35 40 45 gtc ttt gggaaa ggg ctc atc aaa gca gca atg aca act gga gcg tgg 192 Val Phe Gly LysGly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 ata ttc act ggaggg gtt aac aca ggt gtt att cgt cat gtt ggc gat 240 Ile Phe Thr Gly GlyVal Asn Thr Gly Val Ile Arg His Val Gly Asp 65 70 75 80 gcc ttg aag gatcat gcc tct aag tct cga gga aag ata tgc acc ata 288 Ala Leu Lys Asp HisAla Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile 85 90 95 ggt att gcc ccc tgggga att gtg gaa aac cag gag gac ctc att gga 336 Gly Ile Ala Pro Trp GlyIle Val Glu Asn Gln Glu Asp Leu Ile Gly 100 105 110 aga gat gtt gtc cggcca tac cag acc atg tcc aat ccc atg agc aag 384 Arg Asp Val Val Arg ProTyr Gln Thr Met Ser Asn Pro Met Ser Lys 115 120 125 ctc act gtt ctc aacagc atg cat tcc cac ttc att ctg gct gac aac 432 Leu Thr Val Leu Asn SerMet His Ser His Phe Ile Leu Ala Asp Asn 130 135 140 ggg acc act gga aaatat gga gca gag gtg aaa ctt cga aga caa ctg 480 Gly Thr Thr Gly Lys TyrGly Ala Glu Val Lys Leu Arg Arg Gln Leu 145 150 155 160 gaa aag cat atttca ctc cag aag ata aac aca aga atc ggt caa ggt 528 Glu Lys His Ile SerLeu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly 165 170 175 gtt cct gtg gtggca ctc ata gtg gaa gga gga ccc aat gtg atc tcg 576 Val Pro Val Val AlaLeu Ile Val Glu Gly Gly Pro Asn Val Ile Ser 180 185 190 att gtt ttg gagtac ctt cga gac acc cct ccc gtg cca gtg gtt gtc 624 Ile Val Leu Glu TyrLeu Arg Asp Thr Pro Pro Val Pro Val Val Val 195 200 205 tgt gat ggg agtgga cgg gca tcg gac atc ctg gcc ttt ggg cat aaa 672 Cys Asp Gly Ser GlyArg Ala Ser Asp Ile Leu Ala Phe Gly His Lys 210 215 220 tac tca gaa gaaggc gga ctg ata aat gaa tct ttg agg gac cag ctg 720 Tyr Ser Glu Glu GlyGly Leu Ile Asn Glu Ser Leu Arg Asp Gln Leu 225 230 235 240 ttg gtg actata cag aag act ttc aca tac act cga acc caa gct cag 768 Leu Val Thr IleGln Lys Thr Phe Thr Tyr Thr Arg Thr Gln Ala Gln 245 250 255 cat ctg ttcatc atc ctc atg gag tgc atg aag aag aag gaa ttg att 816 His Leu Phe IleIle Leu Met Glu Cys Met Lys Lys Lys Glu Leu Ile 260 265 270 acg gta tttcgg atg gga tca gaa gga cac cag gac att gat ttg gct 864 Thr Val Phe ArgMet Gly Ser Glu Gly His Gln Asp Ile Asp Leu Ala 275 280 285 atc ctg acagct tta ctc aaa gga gcc aat gcc tcg gcc cca gac caa 912 Ile Leu Thr AlaLeu Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp Gln 290 295 300 ctg agc ttagct tta gcc tgg aac aga gtc gac atc gct cgc agc cag 960 Leu Ser Leu AlaLeu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 atc tttatt tac ggg caa cag tgg ccg gtg gga tct ctg gag caa gcc 1008 Ile Phe IleTyr Gly Gln Gln Trp Pro Val Gly Ser Leu Glu Gln Ala 325 330 335 atg ttggat gcc tta gtt ctg gac aga gtg gat ttt gtg aaa tta ctc 1056 Met Leu AspAla Leu Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu 340 345 350 ata gagaat gga gta agc atg cac cgt ttt ctc acc atc tcc aga cta 1104 Ile Glu AsnGly Val Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu 355 360 365 gag gaattg tac aat acg aga cat ggg ccc tca aat aca ttg tac cac 1152 Glu Glu LeuTyr Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His 370 375 380 ttg gtcagg gat gtc aaa aag ggg aac ctg ccc cca gac tac aga atc 1200 Leu Val ArgAsp Val Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile 385 390 395 400 agcctg att gac atc ggc ctg gtg atc gag tac ctg atg ggc ggg gct 1248 Ser LeuIle Asp Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala 405 410 415 tatcgc tgc aac tac acg cgc aag cgc ttc cgg acc ctc tac cac aac 1296 Tyr ArgCys Asn Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn 420 425 430 ctcttc ggc ccc aag agg ccc aaa gcc ttg aaa ctg ctg gga atg gag 1344 Leu PheGly Pro Lys Arg Pro Lys Ala Leu Lys Leu Leu Gly Met Glu 435 440 445 gatgat att ccc ttg agg cga gga aga aag aca acc aag aaa cgt gaa 1392 Asp AspIle Pro Leu Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg Glu 450 455 460 gaagag gtg gac att gac ttg gat gat cct gag atc aac cac ttc ccc 1440 Glu GluVal Asp Ile Asp Leu Asp Asp Pro Glu Ile Asn His Phe Pro 465 470 475 480ttc cct ttc cat gag ctc atg gtg tgg gct gtt ctc atg aag cgg cag 1488 PhePro Phe His Glu Leu Met Val Trp Ala Val Leu Met Lys Arg Gln 485 490 495aag atg gcc ctg ttc ttc tgg cag cac ggt gag gag gcc atg gcc aag 1536 LysMet Ala Leu Phe Phe Trp Gln His Gly Glu Glu Ala Met Ala Lys 500 505 510gcc ctg gtg gcc tgc aag ctc tgc aaa gcc atg gct cat gag gcc tct 1584 AlaLeu Val Ala Cys Lys Leu Cys Lys Ala Met Ala His Glu Ala Ser 515 520 525gag aac gac atg gtt gac gac att tcc cag gag ctg aat cac aat tcc 1632 GluAsn Asp Met Val Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser 530 535 540aga gac ttt ggc cag ctg gct gtg gag ctc ctg gac cag tcc tac aag 1680 ArgAsp Phe Gly Gln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys 545 550 555560 cag gac gaa cag ctg gcc atg aaa ctg ctg acg tat gag ctg aag aac 1728Gln Asp Glu Gln Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn 565 570575 tgg agc aac gcc acg tgc ctg cag ctt gcc gtg gct gcc aaa cac cgc 1776Trp Ser Asn Ala Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His Arg 580 585590 gac ttc atc gcg cac acg tgc agc cag atg ctg ctc acc gac atg tgg 1824Asp Phe Ile Ala His Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp 595 600605 atg ggc cgg ctc cgc atg cgc aag aac tca ggc ctc aag gta att ctg 1872Met Gly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu 610 615620 gga att cta ctt cct cct tca att ctc agc ttg gag ttc aag aac aaa 1920Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys 625 630635 640 gac gac atg ccc tat atg tct cag gcc cag gaa atc cac ctc caa gag1968 Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu 645650 655 aag gag gca gaa gaa cca gag aag ccc aca aag gaa aaa gag gaa gag2016 Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu 660665 670 gac atg gag ctc aca gca atg ttg gga cga aac aac ggg gag tcc tcc2064 Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser 675680 685 agg aag aag gat gaa gag gaa gtt cag agc aag cac cgg tta atc ccc2112 Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His Arg Leu Ile Pro 690695 700 ctc ggc aga aaa atc tat gaa ttc tac aat gca ccc atc gtg aag ttc2160 Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe 705710 715 720 tgg ttc tac aca ctg gcg tat atc gga tac ctg atg ctc ttc aactat 2208 Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr725 730 735 atc gtg tta gtg aag atg gaa cgc tgg ccg tcc acc cag gaa tggatc 2256 Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu Trp Ile740 745 750 gta atc tcc tat att ttc acc ctg gga ata gaa aag atg aga gagatt 2304 Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg Glu Ile755 760 765 ctg atg tca gag cca ggg aag ttg cta cag aaa gtg aag gta tggctg 2352 Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu770 775 780 cag gag tac tgg aat gtc acg gac ctc atc gcc atc ctt ctg ttttct 2400 Gln Glu Tyr Trp Asn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser785 790 795 800 gtc gga atg atc ctt cgt ctc caa gac cag ccc ttc agg agtgac ggg 2448 Val Gly Met Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser AspGly 805 810 815 agg gtc atc tac tgc gtg aac atc att tac tgg tat atc cgtctc cta 2496 Arg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg LeuLeu 820 825 830 gac atc ttc ggc gtg aac aag tat ttg ggc ccg tat gta atgatg att 2544 Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met MetIle 835 840 845 gga aaa atg atg ata gac atg atg tac ttt gtc atc att atgctg gtg 2592 Gly Lys Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met LeuVal 850 855 860 gtt ctg atg agc ttt ggg gtc gcc agg caa gcc atc ctt tttccc aat 2640 Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe ProAsn 865 870 875 880 gag gag cca tca tgg aaa ctg gcc aag aac atc ttc tacatg ccc tat 2688 Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr MetPro Tyr 885 890 895 tgg atg att tat ggg gaa gtg ttt gcg gac cag ata gaccct ccc tgt 2736 Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp ProPro Cys 900 905 910 gga cag aat gag acc cga gag gat ggt aaa ata atc cagctg cct ccc 2784 Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln LeuPro Pro 915 920 925 tgc aag aca gga gct tgg atc gtg ccg gcc atc atg gcctgc tac ctc 2832 Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala CysTyr Leu 930 935 940 tta gtg gca aac atc ttg ctg gtc aac ctc ctc att gctgtc ttt aac 2880 Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala ValPhe Asn 945 950 955 960 aat aca ttt ttt gaa gta aaa tcg ata tcc aac caagtc tgg aag ttt 2928 Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln ValTrp Lys Phe 965 970 975 cag agg tat cag ctc atc atg act ttc cat gaa aggcca gtt ctg ccc 2976 Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg ProVal Leu Pro 980 985 990 cca cca ctg atc atc ttc agc cac atg acc atg atattc cag cac ctg 3024 Pro Pro Leu Ile Ile Phe Ser His Met Thr Met Ile PheGln His Leu 995 1000 1005 tgc tgc cga tgg agg aaa cac gag agc gac ccggat gaa agg gac 3069 Cys Cys Arg Trp Arg Lys His Glu Ser Asp Pro Asp GluArg Asp 1010 1015 1020 tac ggc ctg aaa ctc ttc ata acc gat gat gag ctcaag aaa gta 3114 Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys LysVal 1025 1030 1035 cat gac ttt gaa gag caa tgc ata gaa gaa tac ttc agagaa aag 3159 His Asp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys1040 1045 1050 gat gat cgg ttc aac tca tct aat gat gag agg ata cgg gtgact 3204 Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg Val Thr1055 1060 1065 tca gaa agg gtg gag aac atg tct atg cgg ctg gag gaa gtcaac 3249 Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu Val Asn1070 1075 1080 gag aga gag cac tcc atg aag gct tca ctc cag acc gtg gacatc 3294 Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr Val Asp Ile1085 1090 1095 cgg ctg gcg cag ctg gaa gac ctt atc ggg cgc atg gcc acggcc 3339 Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala1100 1105 1110 ctg gag cgc ctg aca ggt ctg gag cgg gcc gag tcc aac aaaatc 3384 Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile1115 1120 1125 cgc tcg agg acc tcg tca gac tgc acg gac gcc gcc tac attgtc 3429 Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val1130 1135 1140 cgt cag agc agc ttc aac agc cag gaa ggg aac acc ttc aagctc 3474 Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu1145 1150 1155 caa gag agt ata gac cct gca ggt gag gag acc atg tcc ccaact 3519 Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr1160 1165 1170 tct cca acc tta atg ccc cgt atg cga agc cat tct ttc tattca 3564 Ser Pro Thr Leu Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser1175 1180 1185 gtc aat atg aaa gac aaa ggt ggt ata gaa aag ttg gaa agtatt 3609 Val Asn Met Lys Asp Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile1190 1195 1200 ttt aaa gaa agg tcc ctg agc cta cac cgg gct act agt tcccac 3654 Phe Lys Glu Arg Ser Leu Ser Leu His Arg Ala Thr Ser Ser His1205 1210 1215 tct gta gca aaa gaa ccc aaa gct cct gca gcc cct gcc aacacc 3699 Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro Ala Asn Thr1220 1225 1230 ttg gcc att gtt cct gat tcc aga aga cca tca tcg tgt atagac 3744 Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser Ser Cys Ile Asp1235 1240 1245 atc tat gtc tct gct atg gat gag ctc cac tgt gat ata gaccct 3789 Ile Tyr Val Ser Ala Met Asp Glu Leu His Cys Asp Ile Asp Pro1250 1255 1260 ctg gac aat tcc gtg aac atc ctt ggg cta ggc gag cca agcttt 3834 Leu Asp Asn Ser Val Asn Ile Leu Gly Leu Gly Glu Pro Ser Phe1265 1270 1275 tca act cca gta cct tcc aca gcc cct tca agt agt gcc tatgca 3879 Ser Thr Pro Val Pro Ser Thr Ala Pro Ser Ser Ser Ala Tyr Ala1280 1285 1290 aca ctt gca ccc aca gac aga cct cca agc cgg agc att gatttt 3924 Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe1295 1300 1305 gag gac atc acc tcc atg gac act aga tct ttt tct tca gactac 3969 Glu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr1310 1315 1320 acc cac ctc cca gaa tgc caa aac ccc tgg gac tca gag cctccg 4014 Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu Pro Pro1325 1330 1335 atg tac cac acc att gag cgt tcc aaa agt agc cgc tac ctagcc 4059 Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala1340 1345 1350 acc aca ccc ttt ctt cta gaa gag gct ccc att gtg aaa tctcat 4104 Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys Ser His1355 1360 1365 agc ttt atg ttt tcc ccc tca agg agc tat tat gcc aac tttggg 4149 Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly1370 1375 1380 gtg cct gta aaa aca gca gaa tac aca agt att aca gac tgtatt 4194 Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile1385 1390 1395 gac aca agg tgt gtc aat gcc cct caa gca att gcg gac agagct 4239 Asp Thr Arg Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala1400 1405 1410 gcc ttc cct gga ggt ctt gga gac aaa gtg gag gac tta acttgc 4284 Ala Phe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys1415 1420 1425 tgc cat cca gag cga gaa gca gaa ctg agt cac ccc agc tctgac 4329 Cys His Pro Glu Arg Glu Ala Glu Leu Ser His Pro Ser Ser Asp1430 1435 1440 agt gag gag aat gag gcc aaa ggc cgc aga gcc acc att gcaata 4374 Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile1445 1450 1455 tcc tcc cag gag ggt gat aac tca gag aga acc ctg tcc aacaac 4419 Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu Ser Asn Asn1460 1465 1470 atc act gtt ccc aag ata gag cgc gcc aac agc tac tcg gcagag 4464 Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr Ser Ala Glu1475 1480 1485 gag cca agt gcg cca tat gca cac acc agg aag agc ttc tccatc 4509 Glu Pro Ser Ala Pro Tyr Ala His Thr Arg Lys Ser Phe Ser Ile1490 1495 1500 agt gac aaa ctc gac agg cag cgg aac aca gca agc ctg caaaat 4554 Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr Ala Ser Leu Gln Asn1505 1510 1515 ccc ttc cag aga agc aag tcc tcc aag ccg gag ggc cga ggggac 4599 Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro Glu Gly Arg Gly Asp1520 1525 1530 agc ctg tcc atg agg aga ctg tcc aga aca tcg gct ttc caaagc 4644 Ser Leu Ser Met Arg Arg Leu Ser Arg Thr Ser Ala Phe Gln Ser1535 1540 1545 ttt gaa agc aag cac acc taa 4665 Phe Glu Ser Lys His Thr1550 15 1554 PRT Homo sapiens 15 Met Tyr Val Arg Val Ser Phe Asp Thr LysPro Asp Leu Leu Leu His 1 5 10 15 Leu Met Thr Lys Glu Trp Gln Leu GluLeu Pro Lys Leu Leu Ile Ser 20 25 30 Val His Gly Gly Leu Gln Asn Phe GluLeu Gln Pro Lys Leu Lys Gln 35 40 45 Val Phe Gly Lys Gly Leu Ile Lys AlaAla Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe Thr Gly Gly Val Asn Thr GlyVal Ile Arg His Val Gly Asp 65 70 75 80 Ala Leu Lys Asp His Ala Ser LysSer Arg Gly Lys Ile Cys Thr Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile ValGlu Asn Gln Glu Asp Leu Ile Gly 100 105 110 Arg Asp Val Val Arg Pro TyrGln Thr Met Ser Asn Pro Met Ser Lys 115 120 125 Leu Thr Val Leu Asn SerMet His Ser His Phe Ile Leu Ala Asp Asn 130 135 140 Gly Thr Thr Gly LysTyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu 145 150 155 160 Glu Lys HisIle Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly 165 170 175 Val ProVal Val Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser 180 185 190 IleVal Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val Val 195 200 205Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly His Lys 210 215220 Tyr Ser Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu Arg Asp Gln Leu 225230 235 240 Leu Val Thr Ile Gln Lys Thr Phe Thr Tyr Thr Arg Thr Gln AlaGln 245 250 255 His Leu Phe Ile Ile Leu Met Glu Cys Met Lys Lys Lys GluLeu Ile 260 265 270 Thr Val Phe Arg Met Gly Ser Glu Gly His Gln Asp IleAsp Leu Ala 275 280 285 Ile Leu Thr Ala Leu Leu Lys Gly Ala Asn Ala SerAla Pro Asp Gln 290 295 300 Leu Ser Leu Ala Leu Ala Trp Asn Arg Val AspIle Ala Arg Ser Gln 305 310 315 320 Ile Phe Ile Tyr Gly Gln Gln Trp ProVal Gly Ser Leu Glu Gln Ala 325 330 335 Met Leu Asp Ala Leu Val Leu AspArg Val Asp Phe Val Lys Leu Leu 340 345 350 Ile Glu Asn Gly Val Ser MetHis Arg Phe Leu Thr Ile Ser Arg Leu 355 360 365 Glu Glu Leu Tyr Asn ThrArg His Gly Pro Ser Asn Thr Leu Tyr His 370 375 380 Leu Val Arg Asp ValLys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile 385 390 395 400 Ser Leu IleAsp Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala 405 410 415 Tyr ArgCys Asn Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn 420 425 430 LeuPhe Gly Pro Lys Arg Pro Lys Ala Leu Lys Leu Leu Gly Met Glu 435 440 445Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg Glu 450 455460 Glu Glu Val Asp Ile Asp Leu Asp Asp Pro Glu Ile Asn His Phe Pro 465470 475 480 Phe Pro Phe His Glu Leu Met Val Trp Ala Val Leu Met Lys ArgGln 485 490 495 Lys Met Ala Leu Phe Phe Trp Gln His Gly Glu Glu Ala MetAla Lys 500 505 510 Ala Leu Val Ala Cys Lys Leu Cys Lys Ala Met Ala HisGlu Ala Ser 515 520 525 Glu Asn Asp Met Val Asp Asp Ile Ser Gln Glu LeuAsn His Asn Ser 530 535 540 Arg Asp Phe Gly Gln Leu Ala Val Glu Leu LeuAsp Gln Ser Tyr Lys 545 550 555 560 Gln Asp Glu Gln Leu Ala Met Lys LeuLeu Thr Tyr Glu Leu Lys Asn 565 570 575 Trp Ser Asn Ala Thr Cys Leu GlnLeu Ala Val Ala Ala Lys His Arg 580 585 590 Asp Phe Ile Ala His Thr CysSer Gln Met Leu Leu Thr Asp Met Trp 595 600 605 Met Gly Arg Leu Arg MetArg Lys Asn Ser Gly Leu Lys Val Ile Leu 610 615 620 Gly Ile Leu Leu ProPro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys 625 630 635 640 Asp Asp MetPro Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu 645 650 655 Lys GluAla Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu 660 665 670 AspMet Glu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser 675 680 685Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His Arg Leu Ile Pro 690 695700 Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe 705710 715 720 Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met Leu Phe AsnTyr 725 730 735 Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln GluTrp Ile 740 745 750 Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys MetArg Glu Ile 755 760 765 Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys ValLys Val Trp Leu 770 775 780 Gln Glu Tyr Trp Asn Val Thr Asp Leu Ile AlaIle Leu Leu Phe Ser 785 790 795 800 Val Gly Met Ile Leu Arg Leu Gln AspGln Pro Phe Arg Ser Asp Gly 805 810 815 Arg Val Ile Tyr Cys Val Asn IleIle Tyr Trp Tyr Ile Arg Leu Leu 820 825 830 Asp Ile Phe Gly Val Asn LysTyr Leu Gly Pro Tyr Val Met Met Ile 835 840 845 Gly Lys Met Met Ile AspMet Met Tyr Phe Val Ile Ile Met Leu Val 850 855 860 Val Leu Met Ser PheGly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn 865 870 875 880 Glu Glu ProSer Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr 885 890 895 Trp MetIle Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys 900 905 910 GlyGln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro 915 920 925Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys Tyr Leu 930 935940 Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Val Phe Asn 945950 955 960 Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp LysPhe 965 970 975 Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg Pro ValLeu Pro 980 985 990 Pro Pro Leu Ile Ile Phe Ser His Met Thr Met Ile PheGln His Leu 995 1000 1005 Cys Cys Arg Trp Arg Lys His Glu Ser Asp ProAsp Glu Arg Asp 1010 1015 1020 Tyr Gly Leu Lys Leu Phe Ile Thr Asp AspGlu Leu Lys Lys Val 1025 1030 1035 His Asp Phe Glu Glu Gln Cys Ile GluGlu Tyr Phe Arg Glu Lys 1040 1045 1050 Asp Asp Arg Phe Asn Ser Ser AsnAsp Glu Arg Ile Arg Val Thr 1055 1060 1065 Ser Glu Arg Val Glu Asn MetSer Met Arg Leu Glu Glu Val Asn 1070 1075 1080 Glu Arg Glu His Ser MetLys Ala Ser Leu Gln Thr Val Asp Ile 1085 1090 1095 Arg Leu Ala Gln LeuGlu Asp Leu Ile Gly Arg Met Ala Thr Ala 1100 1105 1110 Leu Glu Arg LeuThr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile 1115 1120 1125 Arg Ser ArgThr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val 1130 1135 1140 Arg GlnSer Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu 1145 1150 1155 GlnGlu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr 1160 1165 1170Ser Pro Thr Leu Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser 1175 11801185 Val Asn Met Lys Asp Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile 11901195 1200 Phe Lys Glu Arg Ser Leu Ser Leu His Arg Ala Thr Ser Ser His1205 1210 1215 Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro Ala AsnThr 1220 1225 1230 Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser Ser CysIle Asp 1235 1240 1245 Ile Tyr Val Ser Ala Met Asp Glu Leu His Cys AspIle Asp Pro 1250 1255 1260 Leu Asp Asn Ser Val Asn Ile Leu Gly Leu GlyGlu Pro Ser Phe 1265 1270 1275 Ser Thr Pro Val Pro Ser Thr Ala Pro SerSer Ser Ala Tyr Ala 1280 1285 1290 Thr Leu Ala Pro Thr Asp Arg Pro ProSer Arg Ser Ile Asp Phe 1295 1300 1305 Glu Asp Ile Thr Ser Met Asp ThrArg Ser Phe Ser Ser Asp Tyr 1310 1315 1320 Thr His Leu Pro Glu Cys GlnAsn Pro Trp Asp Ser Glu Pro Pro 1325 1330 1335 Met Tyr His Thr Ile GluArg Ser Lys Ser Ser Arg Tyr Leu Ala 1340 1345 1350 Thr Thr Pro Phe LeuLeu Glu Glu Ala Pro Ile Val Lys Ser His 1355 1360 1365 Ser Phe Met PheSer Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly 1370 1375 1380 Val Pro ValLys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile 1385 1390 1395 Asp ThrArg Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala 1400 1405 1410 AlaPhe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys 1415 1420 1425Cys His Pro Glu Arg Glu Ala Glu Leu Ser His Pro Ser Ser Asp 1430 14351440 Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile 14451450 1455 Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu Ser Asn Asn1460 1465 1470 Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr Ser AlaGlu 1475 1480 1485 Glu Pro Ser Ala Pro Tyr Ala His Thr Arg Lys Ser PheSer Ile 1490 1495 1500 Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr Ala SerLeu Gln Asn 1505 1510 1515 Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro GluGly Arg Gly Asp 1520 1525 1530 Ser Leu Ser Met Arg Arg Leu Ser Arg ThrSer Ala Phe Gln Ser 1535 1540 1545 Phe Glu Ser Lys His Thr 1550 16 8 PRTbacteriophage T7 16 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 17 1566 PRT Homosapiens 17 Met Tyr Val Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu LeuHis 1 5 10 15 Leu Met Thr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu LeuIle Ser 20 25 30 Val His Gly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys LeuLys Gln 35 40 45 Val Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr GlyAla Trp 50 55 60 Ile Phe Thr Gly Gly Val Asn Thr Gly Val Ile Arg His ValGly Asp 65 70 75 80 Ala Leu Lys Asp His Ala Ser Lys Ser Arg Gly Lys IleCys Thr Ile 85 90 95 Gly Ile Ala Pro Trp Gly Ile Val Glu Asn Gln Glu AspLeu Ile Gly 100 105 110 Arg Asp Val Val Arg Pro Tyr Gln Thr Met Ser AsnPro Met Ser Lys 115 120 125 Leu Thr Val Leu Asn Ser Met His Ser His PheIle Leu Ala Asp Asn 130 135 140 Gly Thr Thr Gly Lys Tyr Gly Ala Glu ValLys Leu Arg Arg Gln Leu 145 150 155 160 Glu Lys His Ile Ser Leu Gln LysIle Asn Thr Arg Ile Gly Gln Gly 165 170 175 Val Pro Val Val Ala Leu IleVal Glu Gly Gly Pro Asn Val Ile Ser 180 185 190 Ile Val Leu Glu Tyr LeuArg Asp Thr Pro Pro Val Pro Val Val Val 195 200 205 Cys Asp Gly Ser GlyArg Ala Ser Asp Ile Leu Ala Phe Gly His Lys 210 215 220 Tyr Ser Glu GluGly Gly Leu Ile Asn Glu Ser Leu Arg Asp Gln Leu 225 230 235 240 Leu ValThr Ile Gln Lys Thr Phe Thr Tyr Thr Arg Thr Gln Ala Gln 245 250 255 HisLeu Phe Ile Ile Leu Met Glu Cys Met Lys Lys Lys Glu Leu Ile 260 265 270Thr Val Phe Arg Met Gly Ser Glu Gly His Gln Asp Ile Asp Leu Ala 275 280285 Ile Leu Thr Ala Leu Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp Gln 290295 300 Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln305 310 315 320 Ile Phe Ile Tyr Gly Gln Gln Trp Pro Val Gly Ser Leu GluGln Ala 325 330 335 Met Leu Asp Ala Leu Val Leu Asp Arg Val Asp Phe ValLys Leu Leu 340 345 350 Ile Glu Asn Gly Val Ser Met His Arg Phe Leu ThrIle Ser Arg Leu 355 360 365 Glu Glu Leu Tyr Asn Thr Arg His Gly Pro SerAsn Thr Leu Tyr His 370 375 380 Leu Val Arg Asp Val Lys Lys Arg Glu TyrPro Gly Phe Gly Trp Ile 385 390 395 400 Tyr Phe Lys Gly Asn Leu Pro ProAsp Tyr Arg Ile Ser Leu Ile Asp 405 410 415 Ile Gly Leu Val Ile Glu TyrLeu Met Gly Gly Ala Tyr Arg Cys Asn 420 425 430 Tyr Thr Arg Lys Arg PheArg Thr Leu Tyr His Asn Leu Phe Gly Pro 435 440 445 Lys Arg Pro Lys AlaLeu Lys Leu Leu Gly Met Glu Asp Asp Ile Pro 450 455 460 Leu Arg Arg GlyArg Lys Thr Thr Lys Lys Arg Glu Glu Glu Val Asp 465 470 475 480 Ile AspLeu Asp Asp Pro Glu Ile Asn His Phe Pro Phe Pro Phe His 485 490 495 GluLeu Met Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Leu 500 505 510Phe Phe Trp Gln His Gly Glu Glu Ala Met Ala Lys Ala Leu Val Ala 515 520525 Cys Lys Leu Cys Lys Ala Met Ala His Glu Ala Ser Glu Asn Asp Met 530535 540 Val Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly545 550 555 560 Gln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys Gln AspGlu Gln 565 570 575 Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn TrpSer Asn Ala 580 585 590 Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His ArgAsp Phe Ile Ala 595 600 605 His Thr Cys Ser Gln Met Leu Leu Thr Asp MetTrp Met Gly Arg Leu 610 615 620 Arg Met Arg Lys Asn Ser Gly Leu Lys ValIle Leu Gly Ile Leu Leu 625 630 635 640 Pro Pro Ser Ile Leu Ser Leu GluPhe Lys Asn Lys Asp Asp Met Pro 645 650 655 Tyr Met Ser Gln Ala Gln GluIle His Leu Gln Glu Lys Glu Ala Glu 660 665 670 Glu Pro Glu Lys Pro ThrLys Glu Lys Glu Glu Glu Asp Met Glu Leu 675 680 685 Thr Ala Met Leu GlyArg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp 690 695 700 Glu Glu Glu ValGln Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys 705 710 715 720 Ile TyrGlu Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr 725 730 735 LeuAla Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val 740 745 750Lys Met Glu Arg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr 755 760765 Ile Phe Thr Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu 770775 780 Pro Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp785 790 795 800 Asn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val GlyMet Ile 805 810 815 Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly ArgVal Ile Tyr 820 825 830 Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu LeuAsp Ile Phe Gly 835 840 845 Val Asn Lys Tyr Leu Gly Pro Tyr Val Met MetIle Gly Lys Met Met 850 855 860 Ile Asp Met Met Tyr Phe Val Ile Ile MetLeu Val Val Leu Met Ser 865 870 875 880 Phe Gly Val Ala Arg Gln Ala IleLeu Phe Pro Asn Glu Glu Pro Ser 885 890 895 Trp Lys Leu Ala Lys Asn IlePhe Tyr Met Pro Tyr Trp Met Ile Tyr 900 905 910 Gly Glu Val Phe Ala AspGln Ile Asp Pro Pro Cys Gly Gln Asn Glu 915 920 925 Thr Arg Glu Asp GlyLys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly 930 935 940 Ala Trp Ile ValPro Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn 945 950 955 960 Ile LeuLeu Val Asn Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe 965 970 975 GluVal Lys Ser Ile Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln 980 985 990Leu Ile Met Thr Phe His Glu Arg Pro Val Leu Pro Pro Pro Leu Ile 995 10001005 Ile Phe Ser His Met Thr Met Ile Phe Gln His Leu Cys Cys Arg 10101015 1020 Trp Arg Lys His Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu1025 1030 1035 Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys Lys Val His AspPhe 1040 1045 1050 Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys AspAsp Arg 1055 1060 1065 Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg Val ThrSer Glu Arg 1070 1075 1080 Val Glu Asn Met Ser Met Arg Leu Glu Glu ValAsn Glu Arg Glu 1085 1090 1095 His Ser Met Lys Ala Ser Leu Gln Thr ValAsp Ile Arg Leu Ala 1100 1105 1110 Gln Leu Glu Asp Leu Ile Gly Arg MetAla Thr Ala Leu Glu Arg 1115 1120 1125 Leu Thr Gly Leu Glu Arg Ala GluSer Asn Lys Ile Arg Ser Arg 1130 1135 1140 Thr Ser Ser Asp Cys Thr AspAla Ala Tyr Ile Val Arg Gln Ser 1145 1150 1155 Ser Phe Asn Ser Gln GluGly Asn Thr Phe Lys Leu Gln Glu Ser 1160 1165 1170 Ile Asp Pro Ala GlyGlu Glu Thr Met Ser Pro Thr Ser Pro Thr 1175 1180 1185 Leu Met Pro ArgMet Arg Ser His Ser Phe Tyr Ser Val Asn Met 1190 1195 1200 Lys Asp LysGly Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu 1205 1210 1215 Arg SerLeu Ser Leu His Arg Ala Thr Ser Ser His Ser Val Ala 1220 1225 1230 LysGlu Pro Lys Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile 1235 1240 1245Val Pro Asp Ser Arg Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val 1250 12551260 Ser Ala Met Asp Glu Leu His Cys Asp Ile Asp Pro Leu Asp Asn 12651270 1275 Ser Val Asn Ile Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro1280 1285 1290 Val Pro Ser Thr Ala Pro Ser Ser Ser Ala Tyr Ala Thr LeuAla 1295 1300 1305 Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe GluAsp Ile 1310 1315 1320 Thr Ser Met Asp Thr Arg Ser Phe Ser Ser Asp TyrThr His Leu 1325 1330 1335 Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu ProPro Met Tyr His 1340 1345 1350 Thr Ile Glu Arg Ser Lys Ser Ser Arg TyrLeu Ala Thr Thr Pro 1355 1360 1365 Phe Leu Leu Glu Glu Ala Pro Ile ValLys Ser His Ser Phe Met 1370 1375 1380 Phe Ser Pro Ser Arg Ser Tyr TyrAla Asn Phe Gly Val Pro Val 1385 1390 1395 Lys Thr Ala Glu Tyr Thr SerIle Thr Asp Cys Ile Asp Thr Arg 1400 1405 1410 Cys Val Asn Ala Pro GlnAla Ile Ala Asp Arg Ala Ala Phe Pro 1415 1420 1425 Gly Gly Leu Gly AspLys Val Glu Asp Leu Thr Cys Cys His Pro 1430 1435 1440 Glu Arg Glu AlaGlu Leu Ser His Pro Ser Ser Asp Ser Glu Glu 1445 1450 1455 Asn Glu AlaLys Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln 1460 1465 1470 Glu GlyAsp Asn Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr Val 1475 1480 1485 ProLys Ile Glu Arg Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser 1490 1495 1500Ala Pro Tyr Ala His Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys 1505 15101515 Leu Asp Arg Gln Arg Asn Thr Ala Ser Leu Gln Asn Pro Phe Gln 15201525 1530 Arg Ser Lys Ser Ser Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser1535 1540 1545 Met Arg Arg Leu Ser Arg Thr Ser Ala Phe Gln Ser Phe GluSer 1550 1555 1560 Lys His Thr 1565 18 1566 PRT Homo sapiens 18 Met TyrVal Arg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His 1 5 10 15 LeuMet Thr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 ValHis Gly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln 35 40 45 ValPhe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 IlePhe Thr Gly Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp 65 70 75 80Ala Leu Lys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile 85 90 95Gly Ile Ala Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly 100 105110 Arg Asp Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys 115120 125 Leu Thr Val Leu Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn130 135 140 Gly Thr Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg GlnLeu 145 150 155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg IleGly Gln Gly 165 170 175 Val Pro Val Val Ala Leu Ile Val Glu Gly Gly ProAsn Val Ile Ser 180 185 190 Ile Val Leu Glu Tyr Leu Arg Asp Thr Pro ProVal Pro Val Val Val 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp IleLeu Ala Phe Gly His Lys 210 215 220 Tyr Ser Glu Glu Gly Gly Leu Ile AsnGlu Ser Leu Arg Asp Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys ThrPhe Thr Tyr Thr Arg Thr Gln Ala Gln 245 250 255 His Leu Phe Ile Ile LeuMet Glu Cys Met Lys Lys Lys Glu Leu Ile 260 265 270 Thr Val Phe Arg MetGly Ser Glu Gly His Gln Asp Ile Asp Leu Ala 275 280 285 Ile Leu Thr AlaLeu Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp Gln 290 295 300 Leu Ser LeuAla Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 IlePhe Ile Tyr Gly Gln Gln Trp Pro Val Gly Ser Leu Glu Gln Ala 325 330 335Met Leu Asp Ala Leu Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu 340 345350 Ile Glu Asn Gly Val Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu 355360 365 Glu Glu Leu Tyr Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His370 375 380 Leu Val Arg Asp Val Lys Lys Gly Asn Leu Pro Pro Asp Tyr ArgIle 385 390 395 400 Ser Leu Ile Asp Ile Gly Leu Val Ile Glu Tyr Leu MetGly Gly Ala 405 410 415 Tyr Arg Cys Asn Tyr Thr Arg Lys Arg Phe Arg ThrLeu Tyr His Asn 420 425 430 Leu Phe Gly Pro Lys Arg Pro Lys Ala Leu LysLeu Leu Gly Met Glu 435 440 445 Asp Asp Ile Pro Leu Arg Arg Gly Arg LysThr Thr Lys Lys Arg Glu 450 455 460 Glu Glu Val Asp Ile Asp Leu Asp AspPro Glu Ile Asn His Phe Pro 465 470 475 480 Phe Pro Phe His Glu Leu MetVal Trp Ala Val Leu Met Lys Arg Gln 485 490 495 Lys Met Ala Leu Phe PheTrp Gln His Gly Glu Glu Ala Met Ala Lys 500 505 510 Ala Leu Val Ala CysLys Leu Cys Lys Ala Met Ala His Glu Ala Ser 515 520 525 Glu Asn Asp MetVal Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser 530 535 540 Arg Asp PheGly Gln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys 545 550 555 560 GlnAsp Glu Gln Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn 565 570 575Trp Ser Asn Ala Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His Arg 580 585590 Asp Phe Ile Ala His Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp 595600 605 Met Gly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu610 615 620 Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys AsnLys 625 630 635 640 Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile HisLeu Gln Glu 645 650 655 Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys GluLys Glu Glu Glu 660 665 670 Asp Met Glu Leu Thr Ala Met Leu Gly Arg AsnAsn Gly Glu Ser Ser 675 680 685 Arg Lys Lys Asp Glu Glu Glu Val Gln SerLys His Arg Leu Ile Pro 690 695 700 Leu Gly Arg Lys Ile Tyr Glu Phe TyrAsn Ala Pro Ile Val Lys Phe 705 710 715 720 Trp Phe Tyr Thr Leu Ala TyrIle Gly Tyr Leu Met Leu Phe Asn Tyr 725 730 735 Ile Val Leu Val Lys MetGlu Arg Trp Pro Ser Thr Gln Glu Trp Ile 740 745 750 Val Ile Ser Tyr IlePhe Thr Leu Gly Ile Glu Lys Met Arg Glu Ile 755 760 765 Leu Met Ser GluPro Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu 770 775 780 Gln Glu TyrTrp Asn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser 785 790 795 800 ValGly Met Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly 805 810 815Arg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 820 825830 Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile 835840 845 Gly Lys Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val850 855 860 Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe ProAsn 865 870 875 880 Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe TyrMet Pro Tyr 885 890 895 Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln IleAsp Arg Lys Gln 900 905 910 Val Tyr Asp Ser His Thr Pro Lys Ser Ala ProCys Gly Gln Asn Glu 915 920 925 Thr Arg Glu Asp Gly Lys Ile Ile Gln LeuPro Pro Cys Lys Thr Gly 930 935 940 Ala Trp Ile Val Pro Ala Ile Met AlaCys Tyr Leu Leu Val Ala Asn 945 950 955 960 Ile Leu Leu Val Asn Leu LeuIle Ala Val Phe Asn Asn Thr Phe Phe 965 970 975 Glu Val Lys Ser Ile SerAsn Gln Val Trp Lys Phe Gln Arg Tyr Gln 980 985 990 Leu Ile Met Thr PheHis Glu Arg Pro Val Leu Pro Pro Pro Leu Ile 995 1000 1005 Ile Phe SerHis Met Thr Met Ile Phe Gln His Leu Cys Cys Arg 1010 1015 1020 Trp ArgLys His Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu 1025 1030 1035 LysLeu Phe Ile Thr Asp Asp Glu Leu Lys Lys Val His Asp Phe 1040 1045 1050Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys Asp Asp Arg 1055 10601065 Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg Val Thr Ser Glu Arg 10701075 1080 Val Glu Asn Met Ser Met Arg Leu Glu Glu Val Asn Glu Arg Glu1085 1090 1095 His Ser Met Lys Ala Ser Leu Gln Thr Val Asp Ile Arg LeuAla 1100 1105 1110 Gln Leu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala LeuGlu Arg 1115 1120 1125 Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys IleArg Ser Arg 1130 1135 1140 Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr IleVal Arg Gln Ser 1145 1150 1155 Ser Phe Asn Ser Gln Glu Gly Asn Thr PheLys Leu Gln Glu Ser 1160 1165 1170 Ile Asp Pro Ala Gly Glu Glu Thr MetSer Pro Thr Ser Pro Thr 1175 1180 1185 Leu Met Pro Arg Met Arg Ser HisSer Phe Tyr Ser Val Asn Met 1190 1195 1200 Lys Asp Lys Gly Gly Ile GluLys Leu Glu Ser Ile Phe Lys Glu 1205 1210 1215 Arg Ser Leu Ser Leu HisArg Ala Thr Ser Ser His Ser Val Ala 1220 1225 1230 Lys Glu Pro Lys AlaPro Ala Ala Pro Ala Asn Thr Leu Ala Ile 1235 1240 1245 Val Pro Asp SerArg Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val 1250 1255 1260 Ser Ala MetAsp Glu Leu His Cys Asp Ile Asp Pro Leu Asp Asn 1265 1270 1275 Ser ValAsn Ile Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro 1280 1285 1290 ValPro Ser Thr Ala Pro Ser Ser Ser Ala Tyr Ala Thr Leu Ala 1295 1300 1305Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe Glu Asp Ile 1310 13151320 Thr Ser Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr Thr His Leu 13251330 1335 Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu Pro Pro Met Tyr His1340 1345 1350 Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala Thr ThrPro 1355 1360 1365 Phe Leu Leu Glu Glu Ala Pro Ile Val Lys Ser His SerPhe Met 1370 1375 1380 Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe GlyVal Pro Val 1385 1390 1395 Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp CysIle Asp Thr Arg 1400 1405 1410 Cys Val Asn Ala Pro Gln Ala Ile Ala AspArg Ala Ala Phe Pro 1415 1420 1425 Gly Gly Leu Gly Asp Lys Val Glu AspLeu Thr Cys Cys His Pro 1430 1435 1440 Glu Arg Glu Ala Glu Leu Ser HisPro Ser Ser Asp Ser Glu Glu 1445 1450 1455 Asn Glu Ala Lys Gly Arg ArgAla Thr Ile Ala Ile Ser Ser Gln 1460 1465 1470 Glu Gly Asp Asn Ser GluArg Thr Leu Ser Asn Asn Ile Thr Val 1475 1480 1485 Pro Lys Ile Glu ArgAla Asn Ser Tyr Ser Ala Glu Glu Pro Ser 1490 1495 1500 Ala Pro Tyr AlaHis Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys 1505 1510 1515 Leu Asp ArgGln Arg Asn Thr Ala Ser Leu Gln Asn Pro Phe Gln 1520 1525 1530 Arg SerLys Ser Ser Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser 1535 1540 1545 MetArg Arg Leu Ser Arg Thr Ser Ala Phe Gln Ser Phe Glu Ser 1550 1555 1560Lys His Thr 1565 19 1544 PRT Homo sapiens 19 Met Tyr Val Arg Val Ser PheAsp Thr Lys Pro Asp Leu Leu Leu His 1 5 10 15 Leu Met Thr Lys Glu TrpGln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His Gly Gly Leu GlnAsn Phe Glu Leu Gln Pro Lys Leu Lys Gln 35 40 45 Val Phe Gly Lys Gly LeuIle Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe Thr Gly Gly ValAsn Thr Gly Val Ile Arg His Val Gly Asp 65 70 75 80 Ala Leu Lys Asp HisAla Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile 85 90 95 Gly Ile Ala Pro TrpGly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly 100 105 110 Arg Asp Val ValArg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys 115 120 125 Leu Thr ValLeu Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn 130 135 140 Gly ThrThr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu 145 150 155 160Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly 165 170175 Val Pro Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser 180185 190 Ile Val Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val Val195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly HisLys 210 215 220 Tyr Ser Glu Glu Gly Gly Leu Ile Asn Glu Ser Leu Arg AspGln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe Thr Tyr Thr ArgThr Gln Ala Gln 245 250 255 His Leu Phe Ile Ile Leu Met Glu Cys Met LysLys Lys Glu Leu Ile 260 265 270 Thr Val Phe Arg Met Gly Ser Glu Gly HisGln Asp Ile Asp Leu Ala 275 280 285 Ile Leu Thr Ala Leu Leu Lys Gly AlaAsn Ala Ser Ala Pro Asp Gln 290 295 300 Leu Ser Leu Ala Leu Ala Trp AsnArg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 Ile Phe Ile Tyr Gly GlnGln Trp Pro Val Gly Ser Leu Glu Gln Ala 325 330 335 Met Leu Asp Ala LeuVal Leu Asp Arg Val Asp Phe Val Lys Leu Leu 340 345 350 Ile Glu Asn GlyVal Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu 355 360 365 Glu Glu LeuTyr Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His 370 375 380 Leu ValArg Asp Val Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile 385 390 395 400Ser Leu Ile Asp Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala 405 410415 Tyr Arg Cys Asn Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn 420425 430 Leu Phe Gly Pro Lys Arg Asp Asp Ile Pro Leu Arg Arg Gly Arg Lys435 440 445 Thr Thr Lys Lys Arg Glu Glu Glu Val Asp Ile Asp Leu Asp AspPro 450 455 460 Glu Ile Asn His Phe Pro Phe Pro Phe His Glu Leu Met ValTrp Ala 465 470 475 480 Val Leu Met Lys Arg Gln Lys Met Ala Leu Phe PheTrp Gln His Gly 485 490 495 Glu Glu Ala Met Ala Lys Ala Leu Val Ala CysLys Leu Cys Lys Ala 500 505 510 Met Ala His Glu Ala Ser Glu Asn Asp MetVal Asp Asp Ile Ser Gln 515 520 525 Glu Leu Asn His Asn Ser Arg Asp PheGly Gln Leu Ala Val Glu Leu 530 535 540 Leu Asp Gln Ser Tyr Lys Gln AspGlu Gln Leu Ala Met Lys Leu Leu 545 550 555 560 Thr Tyr Glu Leu Lys AsnTrp Ser Asn Ala Thr Cys Leu Gln Leu Ala 565 570 575 Val Ala Ala Lys HisArg Asp Phe Ile Ala His Thr Cys Ser Gln Met 580 585 590 Leu Leu Thr AspMet Trp Met Gly Arg Leu Arg Met Arg Lys Asn Ser 595 600 605 Gly Leu LysVal Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser 610 615 620 Leu GluPhe Lys Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln 625 630 635 640Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr 645 650655 Lys Glu Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg 660665 670 Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser675 680 685 Lys His Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe TyrAsn 690 695 700 Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr IleGly Tyr 705 710 715 720 Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys MetGlu Arg Trp Pro 725 730 735 Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr IlePhe Thr Leu Gly Ile 740 745 750 Glu Lys Met Arg Glu Ile Leu Met Ser GluPro Gly Lys Leu Leu Gln 755 760 765 Lys Val Lys Val Trp Leu Gln Glu TyrTrp Asn Val Thr Asp Leu Ile 770 775 780 Ala Ile Leu Leu Phe Ser Val GlyMet Ile Leu Arg Leu Gln Asp Gln 785 790 795 800 Pro Phe Arg Ser Asp GlyArg Val Ile Tyr Cys Val Asn Ile Ile Tyr 805 810 815 Trp Tyr Ile Arg LeuLeu Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly 820 825 830 Pro Tyr Val MetMet Ile Gly Lys Met Met Ile Asp Met Met Tyr Phe 835 840 845 Val Ile IleMet Leu Val Val Leu Met Ser Phe Gly Val Ala Arg Gln 850 855 860 Ala IleLeu Phe Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn 865 870 875 880Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp 885 890895 Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys 900905 910 Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala915 920 925 Ile Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val AsnLeu 930 935 940 Leu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu Val Lys SerIle Ser 945 950 955 960 Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu IleMet Thr Phe His 965 970 975 Glu Arg Pro Val Leu Pro Pro Pro Leu Ile IlePhe Ser His Met Thr 980 985 990 Met Ile Phe Gln His Leu Cys Cys Arg TrpArg Lys His Glu Ser Asp 995 1000 1005 Pro Asp Glu Arg Asp Tyr Gly LeuLys Leu Phe Ile Thr Asp Asp 1010 1015 1020 Glu Leu Lys Lys Val His AspPhe Glu Glu Gln Cys Ile Glu Glu 1025 1030 1035 Tyr Phe Arg Glu Lys AspAsp Arg Phe Asn Ser Ser Asn Asp Glu 1040 1045 1050 Arg Ile Arg Val ThrSer Glu Arg Val Glu Asn Met Ser Met Arg 1055 1060 1065 Leu Glu Glu ValAsn Glu Arg Glu His Ser Met Lys Ala Ser Leu 1070 1075 1080 Gln Thr ValAsp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly 1085 1090 1095 Arg MetAla Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala 1100 1105 1110 GluSer Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp 1115 1120 1125Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly 1130 11351140 Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu 11451150 1155 Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg Ser1160 1165 1170 His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly IleGlu 1175 1180 1185 Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser LeuHis Arg 1190 1195 1200 Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro LysAla Pro Ala 1205 1210 1215 Ala Pro Ala Asn Thr Leu Ala Ile Val Pro AspSer Arg Arg Pro 1220 1225 1230 Ser Ser Cys Ile Asp Ile Tyr Val Ser AlaMet Asp Glu Leu His 1235 1240 1245 Cys Asp Ile Asp Pro Leu Asp Asn SerVal Asn Ile Leu Gly Leu 1250 1255 1260 Gly Glu Pro Ser Phe Ser Thr ProVal Pro Ser Thr Ala Pro Ser 1265 1270 1275 Ser Ser Ala Tyr Ala Thr LeuAla Pro Thr Asp Arg Pro Pro Ser 1280 1285 1290 Arg Ser Ile Asp Phe GluAsp Ile Thr Ser Met Asp Thr Arg Ser 1295 1300 1305 Phe Ser Ser Asp TyrThr His Leu Pro Glu Cys Gln Asn Pro Trp 1310 1315 1320 Asp Ser Glu ProPro Met Tyr His Thr Ile Glu Arg Ser Lys Ser 1325 1330 1335 Ser Arg TyrLeu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro 1340 1345 1350 Ile ValLys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr 1355 1360 1365 TyrAla Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser 1370 1375 1380Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala 1385 13901395 Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys Val 14001405 1410 Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu Ser1415 1420 1425 His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly ArgArg 1430 1435 1440 Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn SerGlu Arg 1445 1450 1455 Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile GluArg Ala Asn 1460 1465 1470 Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro TyrAla His Thr Arg 1475 1480 1485 Lys Ser Phe Ser Ile Ser Asp Lys Leu AspArg Gln Arg Asn Thr 1490 1495 1500 Ala Ser Leu Gln Asn Pro Phe Gln ArgSer Lys Ser Ser Lys Pro 1505 1510 1515 Glu Gly Arg Gly Asp Ser Leu SerMet Arg Arg Leu Ser Arg Thr 1520 1525 1530 Ser Ala Phe Gln Ser Phe GluSer Lys His Thr 1535 1540 20 1556 PRT Homo sapiens 20 Met Tyr Val ArgVal Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His 1 5 10 15 Leu Met ThrLys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val His GlyGly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln 35 40 45 Val Phe GlyLys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile Phe ThrGly Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp 65 70 75 80 Ala LeuLys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile 85 90 95 Gly IleAla Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly 100 105 110 ArgAsp Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys 115 120 125Leu Thr Val Leu Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn 130 135140 Gly Thr Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu 145150 155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly GlnGly 165 170 175 Val Pro Val Val Ala Leu Ile Val Glu Gly Gly Pro Asn ValIle Ser 180 185 190 Ile Val Leu Glu Tyr Leu Arg Asp Thr Pro Pro Val ProVal Val Val 195 200 205 Cys Asp Gly Ser Gly Arg Ala Ser Asp Ile Leu AlaPhe Gly His Lys 210 215 220 Tyr Ser Glu Glu Gly Gly Leu Ile Asn Glu SerLeu Arg Asp Gln Leu 225 230 235 240 Leu Val Thr Ile Gln Lys Thr Phe ThrTyr Thr Arg Thr Gln Ala Gln 245 250 255 His Leu Phe Ile Ile Leu Met GluCys Met Lys Lys Lys Glu Leu Ile 260 265 270 Thr Val Phe Arg Met Gly SerGlu Gly His Gln Asp Ile Asp Leu Ala 275 280 285 Ile Leu Thr Ala Leu LeuLys Gly Ala Asn Ala Ser Ala Pro Asp Gln 290 295 300 Leu Ser Leu Ala LeuAla Trp Asn Arg Val Asp Ile Ala Arg Ser Gln 305 310 315 320 Ile Phe IleTyr Gly Gln Gln Trp Pro Val Gly Ser Leu Glu Gln Ala 325 330 335 Met LeuAsp Ala Leu Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu 340 345 350 IleGlu Asn Gly Val Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu 355 360 365Glu Glu Leu Tyr Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His 370 375380 Leu Val Arg Asp Val Lys Lys Arg Glu Tyr Pro Gly Phe Gly Trp Ile 385390 395 400 Tyr Phe Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu IleAsp 405 410 415 Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr ArgCys Asn 420 425 430 Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn LeuPhe Gly Pro 435 440 445 Lys Arg Asp Asp Ile Pro Leu Arg Arg Gly Arg LysThr Thr Lys Lys 450 455 460 Arg Glu Glu Glu Val Asp Ile Asp Leu Asp AspPro Glu Ile Asn His 465 470 475 480 Phe Pro Phe Pro Phe His Glu Leu MetVal Trp Ala Val Leu Met Lys 485 490 495 Arg Gln Lys Met Ala Leu Phe PheTrp Gln His Gly Glu Glu Ala Met 500 505 510 Ala Lys Ala Leu Val Ala CysLys Leu Cys Lys Ala Met Ala His Glu 515 520 525 Ala Ser Glu Asn Asp MetVal Asp Asp Ile Ser Gln Glu Leu Asn His 530 535 540 Asn Ser Arg Asp PheGly Gln Leu Ala Val Glu Leu Leu Asp Gln Ser 545 550 555 560 Tyr Lys GlnAsp Glu Gln Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu 565 570 575 Lys AsnTrp Ser Asn Ala Thr Cys Leu Gln Leu Ala Val Ala Ala Lys 580 585 590 HisArg Asp Phe Ile Ala His Thr Cys Ser Gln Met Leu Leu Thr Asp 595 600 605Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn Ser Gly Leu Lys Val 610 615620 Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys 625630 635 640 Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile HisLeu 645 650 655 Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys GluLys Glu 660 665 670 Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg AsnAsn Gly Glu 675 680 685 Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln SerLys His Arg Leu 690 695 700 Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe TyrAsn Ala Pro Ile Val 705 710 715 720 Lys Phe Trp Phe Tyr Thr Leu Ala TyrIle Gly Tyr Leu Met Leu Phe 725 730 735 Asn Tyr Ile Val Leu Val Lys MetGlu Arg Trp Pro Ser Thr Gln Glu 740 745 750 Trp Ile Val Ile Ser Tyr IlePhe Thr Leu Gly Ile Glu Lys Met Arg 755 760 765 Glu Ile Leu Met Ser GluPro Gly Lys Leu Leu Gln Lys Val Lys Val 770 775 780 Trp Leu Gln Glu TyrTrp Asn Val Thr Asp Leu Ile Ala Ile Leu Leu 785 790 795 800 Phe Ser ValGly Met Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser 805 810 815 Asp GlyArg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg 820 825 830 LeuLeu Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met 835 840 845Met Ile Gly Lys Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met 850 855860 Leu Val Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe 865870 875 880 Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe TyrMet 885 890 895 Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln IleAsp Pro 900 905 910 Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys IleIle Gln Leu 915 920 925 Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro AlaIle Met Ala Cys 930 935 940 Tyr Leu Leu Val Ala Asn Ile Leu Leu Val AsnLeu Leu Ile Ala Val 945 950 955 960 Phe Asn Asn Thr Phe Phe Glu Val LysSer Ile Ser Asn Gln Val Trp 965 970 975 Lys Phe Gln Arg Tyr Gln Leu IleMet Thr Phe His Glu Arg Pro Val 980 985 990 Leu Pro Pro Pro Leu Ile IlePhe Ser His Met Thr Met Ile Phe Gln 995 1000 1005 His Leu Cys Cys ArgTrp Arg Lys His Glu Ser Asp Pro Asp Glu 1010 1015 1020 Arg Asp Tyr GlyLeu Lys Leu Phe Ile Thr Asp Asp Glu Leu Lys 1025 1030 1035 Lys Val HisAsp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg 1040 1045 1050 Glu LysAsp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg 1055 1060 1065 ValThr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu 1070 1075 1080Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr Val 1085 10901095 Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala 11001105 1110 Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn1115 1120 1125 Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala AlaTyr 1130 1135 1140 Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly AsnThr Phe 1145 1150 1155 Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu GluThr Met Ser 1160 1165 1170 Pro Thr Ser Pro Thr Leu Met Pro Arg Met ArgSer His Ser Phe 1175 1180 1185 Tyr Ser Val Asn Met Lys Asp Lys Gly GlyIle Glu Lys Leu Glu 1190 1195 1200 Ser Ile Phe Lys Glu Arg Ser Leu SerLeu His Arg Ala Thr Ser 1205 1210 1215 Ser His Ser Val Ala Lys Glu ProLys Ala Pro Ala Ala Pro Ala 1220 1225 1230 Asn Thr Leu Ala Ile Val ProAsp Ser Arg Arg Pro Ser Ser Cys 1235 1240 1245 Ile Asp Ile Tyr Val SerAla Met Asp Glu Leu His Cys Asp Ile 1250 1255 1260 Asp Pro Leu Asp AsnSer Val Asn Ile Leu Gly Leu Gly Glu Pro 1265 1270 1275 Ser Phe Ser ThrPro Val Pro Ser Thr Ala Pro Ser Ser Ser Ala 1280 1285 1290 Tyr Ala ThrLeu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile 1295 1300 1305 Asp PheGlu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser 1310 1315 1320 AspTyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu 1325 1330 1335Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr 1340 13451350 Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys 13551360 1365 Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn1370 1375 1380 Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile ThrAsp 1385 1390 1395 Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala IleAla Asp 1400 1405 1410 Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys ValGlu Asp Leu 1415 1420 1425 Thr Cys Cys His Pro Glu Arg Glu Ala Glu LeuSer His Pro Ser 1430 1435 1440 Ser Asp Ser Glu Glu Asn Glu Ala Lys GlyArg Arg Ala Thr Ile 1445 1450 1455 Ala Ile Ser Ser Gln Glu Gly Asp AsnSer Glu Arg Thr Leu Ser 1460 1465 1470 Asn Asn Ile Thr Val Pro Lys IleGlu Arg Ala Asn Ser Tyr Ser 1475 1480 1485 Ala Glu Glu Pro Ser Ala ProTyr Ala His Thr Arg Lys Ser Phe 1490 1495 1500 Ser Ile Ser Asp Lys LeuAsp Arg Gln Arg Asn Thr Ala Ser Leu 1505 1510 1515 Gln Asn Pro Phe GlnArg Ser Lys Ser Ser Lys Pro Glu Gly Arg 1520 1525 1530 Gly Asp Ser LeuSer Met Arg Arg Leu Ser Arg Thr Ser Ala Phe 1535 1540 1545 Gln Ser PheGlu Ser Lys His Thr 1550 1555 21 1579 PRT Homo sapiens 21 Met Tyr ValArg Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His 1 5 10 15 Leu MetThr Lys Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser 20 25 30 Val HisGly Gly Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln 35 40 45 Val PheGly Lys Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp 50 55 60 Ile PheThr Gly Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp 65 70 75 80 AlaLeu Lys Asp His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile 85 90 95 GlyIle Ala Pro Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly 100 105 110Arg Asp Val Val Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys 115 120125 Leu Thr Val Leu Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn 130135 140 Gly Thr Thr Gly Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu145 150 155 160 Glu Lys His Ile Ser Leu Gln Lys Ile Asn Thr Arg Cys LeuPro Phe 165 170 175 Phe Ser Leu Asp Ser Arg Leu Phe Tyr Ser Phe Trp GlySer Cys Gln 180 185 190 Leu Asp Ser Val Gly Ile Gly Gln Gly Val Pro ValVal Ala Leu Ile 195 200 205 Val Glu Gly Gly Pro Asn Val Ile Ser Ile ValLeu Glu Tyr Leu Arg 210 215 220 Asp Thr Pro Pro Val Pro Val Val Val CysAsp Gly Ser Gly Arg Ala 225 230 235 240 Ser Asp Ile Leu Ala Phe Gly HisLys Tyr Ser Glu Glu Gly Gly Leu 245 250 255 Ile Asn Glu Ser Leu Arg AspGln Leu Leu Val Thr Ile Gln Lys Thr 260 265 270 Phe Thr Tyr Thr Arg ThrGln Ala Gln His Leu Phe Ile Ile Leu Met 275 280 285 Glu Cys Met Lys LysLys Glu Leu Ile Thr Val Phe Arg Met Gly Ser 290 295 300 Glu Gly His GlnAsp Ile Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys 305 310 315 320 Gly AlaAsn Ala Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu Ala Trp 325 330 335 AsnArg Val Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly Gln Gln 340 345 350Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu Val Leu 355 360365 Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Ser Met 370375 380 His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr Asn Thr Arg385 390 395 400 His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp ValLys Lys 405 410 415 Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile AspIle Gly Leu 420 425 430 Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg CysAsn Tyr Thr Arg 435 440 445 Lys Arg Phe Arg Thr Leu Tyr His Asn Leu PheGly Pro Lys Arg Pro 450 455 460 Lys Ala Leu Lys Leu Leu Gly Met Glu AspAsp Ile Pro Leu Arg Arg 465 470 475 480 Gly Arg Lys Thr Thr Lys Lys ArgGlu Glu Glu Val Asp Ile Asp Leu 485 490 495 Asp Asp Pro Glu Ile Asn HisPhe Pro Phe Pro Phe His Glu Leu Met 500 505 510 Val Trp Ala Val Leu MetLys Arg Gln Lys Met Ala Leu Phe Phe Trp 515 520 525 Gln His Gly Glu GluAla Met Ala Lys Ala Leu Val Ala Cys Lys Leu 530 535 540 Cys Lys Ala MetAla His Glu Ala Ser Glu Asn Asp Met Val Asp Asp 545 550 555 560 Ile SerGln Glu Leu Asn His Asn Ser Arg Asp Phe Gly Gln Leu Ala 565 570 575 ValGlu Leu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu Ala Met 580 585 590Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys Leu 595 600605 Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr Cys 610615 620 Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg625 630 635 640 Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu ProPro Ser 645 650 655 Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met ProTyr Met Ser 660 665 670 Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu AlaGlu Glu Pro Glu 675 680 685 Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp MetGlu Leu Thr Ala Met 690 695 700 Leu Gly Arg Asn Asn Gly Glu Ser Ser ArgLys Lys Asp Glu Glu Glu 705 710 715 720 Val Gln Ser Lys His Arg Leu IlePro Leu Gly Arg Lys Ile Tyr Glu 725 730 735 Phe Tyr Asn Ala Pro Ile ValLys Phe Trp Phe Tyr Thr Leu Ala Tyr 740 745 750 Ile Gly Tyr Leu Met LeuPhe Asn Tyr Ile Val Leu Val Lys Met Glu 755 760 765 Arg Trp Pro Ser ThrGln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr 770 775 780 Leu Gly Ile GluLys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys 785 790 795 800 Leu LeuGln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr 805 810 815 AspLeu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu 820 825 830Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn 835 840845 Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys 850855 860 Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met865 870 875 880 Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser PheGly Val 885 890 895 Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro SerTrp Lys Leu 900 905 910 Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met IleTyr Gly Glu Val 915 920 925 Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly GlnAsn Glu Thr Arg Glu 930 935 940 Asp Gly Lys Ile Ile Gln Leu Pro Pro CysLys Thr Gly Ala Trp Ile 945 950 955 960 Val Pro Ala Ile Met Ala Cys TyrLeu Leu Val Ala Asn Ile Leu Leu 965 970 975 Val Asn Leu Leu Ile Ala ValPhe Asn Asn Thr Phe Phe Glu Val Lys 980 985 990 Ser Ile Ser Asn Gln ValTrp Lys Phe Gln Arg Tyr Gln Leu Ile Met 995 1000 1005 Thr Phe His GluArg Pro Val Leu Pro Pro Pro Leu Ile Ile Phe 1010 1015 1020 Ser His MetThr Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg 1025 1030 1035 Lys HisGlu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu 1040 1045 1050 PheIle Thr Asp Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu 1055 1060 1065Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn 1070 10751080 Ser Ser Asn Asp Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu 10851090 1095 Asn Met Ser Met Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser1100 1105 1110 Met Lys Ala Ser Leu Gln Thr Val Asp Ile Arg Leu Ala GlnLeu 1115 1120 1125 Glu Asp Leu Ile Gly Arg Met Ala Thr Ala Leu Glu ArgLeu Thr 1130 1135 1140 Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg SerArg Thr Ser 1145 1150 1155 Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val ArgGln Ser Ser Phe 1160 1165 1170 Asn Ser Gln Glu Gly Asn Thr Phe Lys LeuGln Glu Ser Ile Asp 1175 1180 1185 Pro Ala Gly Glu Glu Thr Met Ser ProThr Ser Pro Thr Leu Met 1190 1195 1200 Pro Arg Met Arg Ser His Ser PheTyr Ser Val Asn Met Lys Asp 1205 1210 1215 Lys Gly Gly Ile Glu Lys LeuGlu Ser Ile Phe Lys Glu Arg Ser 1220 1225 1230 Leu Ser Leu His Arg AlaThr Ser Ser His Ser Val Ala Lys Glu 1235 1240 1245 Pro Lys Ala Pro AlaAla Pro Ala Asn Thr Leu Ala Ile Val Pro 1250 1255 1260 Asp Ser Arg ArgPro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala 1265 1270 1275 Met Asp GluLeu His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val 1280 1285 1290 Asn IleLeu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro 1295 1300 1305 SerThr Ala Pro Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr 1310 1315 1320Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser 1325 13301335 Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu 13401345 1350 Cys Gln Asn Pro Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile1355 1360 1365 Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr Pro PheLeu 1370 1375 1380 Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser Phe MetPhe Ser 1385 1390 1395 Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val ProVal Lys Thr 1400 1405 1410 Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile AspThr Arg Cys Val 1415 1420 1425 Asn Ala Pro Gln Ala Ile Ala Asp Arg AlaAla Phe Pro Gly Gly 1430 1435 1440 Leu Gly Asp Lys Val Glu Asp Leu ThrCys Cys His Pro Glu Arg 1445 1450 1455 Glu Ala Glu Leu Ser His Pro SerSer Asp Ser Glu Glu Asn Glu 1460 1465 1470 Ala Lys Gly Arg Arg Ala ThrIle Ala Ile Ser Ser Gln Glu Gly 1475 1480 1485 Asp Asn Ser Glu Arg ThrLeu Ser Asn Asn Ile Thr Val Pro Lys 1490 1495 1500 Ile Glu Arg Ala AsnSer Tyr Ser Ala Glu Glu Pro Ser Ala Pro 1505 1510 1515 Tyr Ala His ThrArg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp 1520 1525 1530 Arg Gln ArgAsn Thr Ala Ser Leu Gln Asn Pro Phe Gln Arg Ser 1535 1540 1545 Lys SerSer Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg 1550 1555 1560 ArgLeu Ser Arg Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His 1565 1570 1575Thr 22 18 PRT Homo sapiens 22 Leu Lys Val Ile Leu Gly Ile Leu Leu ProPro Ser Ile Leu Ser Leu 1 5 10 15 Glu Phe 23 18 PRT Homo sapiens 23 IleVal Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met 1 5 10 15Leu Phe 24 14 PRT Homo sapiens 24 Val Thr Asp Leu Ile Ala Ile Leu LeuPhe Ser Val Gly Met 1 5 10 25 18 PRT Homo sapiens 25 Arg Val Ile Tyr CysVal Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 1 5 10 15 Asp Ile 26 18 PRTHomo sapiens 26 Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu ValVal Leu 1 5 10 15 Met Ser 27 21 PRT Homo sapiens 27 Ala Ile Met Ala CysTyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn 1 5 10 15 Leu Leu Ile AlaVal 20 28 79 PRT Homo sapiens 28 Lys Asn Lys Asp Asp Met Pro Tyr Met SerGln Ala Gln Glu Ile His 1 5 10 15 Leu Gln Glu Lys Glu Ala Glu Glu ProGlu Lys Pro Thr Lys Glu Lys 20 25 30 Glu Glu Glu Asp Met Glu Leu Thr AlaMet Leu Gly Arg Asn Asn Gly 35 40 45 Glu Ser Ser Arg Lys Lys Asp Glu GluGlu Val Gln Ser Lys His Arg 50 55 60 Leu Ile Pro Leu Gly Arg Lys Ile TyrGlu Phe Tyr Asn Ala Pro 65 70 75 29 55 PRT Homo sapiens 29 Asn Tyr IleVal Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu 1 5 10 15 Trp IleVal Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg 20 25 30 Glu IleLeu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys Val 35 40 45 Trp LeuGln Glu Tyr Trp Asn 50 55 30 13 PRT Homo sapiens 30 Ile Leu Arg Leu GlnAsp Gln Pro Phe Arg Ser Asp Gly 1 5 10 31 16 PRT Homo sapiens 31 Phe GlyVal Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys 1 5 10 15 32 69PRT Homo sapiens 32 Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn GluGlu Pro Ser 1 5 10 15 Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro TyrTrp Met Ile Tyr 20 25 30 Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro CysGly Gln Asn Glu 35 40 45 Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro ProCys Lys Thr Gly 50 55 60 Ala Trp Ile Val Pro 65 33 6 PRT Homo sapiens 33Val Trp Lys Phe Gln Arg 1 5 34 212 PRT Homo sapiens 34 Thr Gln Glu TrpIle Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu 1 5 10 15 Lys Met ArgGlu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys 20 25 30 Val Lys ValTrp Leu Gln Glu Tyr Trp Asn Val Thr Asp Leu Ile Ala 35 40 45 Ile Leu LeuPhe Ser Val Gly Met Ile Leu Arg Leu Gln Asp Gln Pro 50 55 60 Phe Arg SerAsp Gly Arg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp 65 70 75 80 Tyr IleArg Leu Leu Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly Pro 85 90 95 Tyr ValMet Met Ile Gly Lys Met Met Ile Asp Met Met Tyr Phe Val 100 105 110 IleIle Met Leu Val Val Leu Met Ser Phe Gly Val Ala Arg Gln Ala 115 120 125Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile 130 135140 Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln 145150 155 160 Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly LysIle 165 170 175 Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val ProAla Ile 180 185 190 Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu ValAsn Leu Leu 195 200 205 Ile Ala Val Phe 210 35 56 PRT Homo sapiens 35Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg 1 5 1015 Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln 20 2530 Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met 35 4045 Ala Thr Ala Leu Glu Arg Leu Thr 50 55 36 18 PRT Homo sapiens 36 LeuLys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu Ser Leu 1 5 10 15Glu Phe 37 18 PRT Homo sapiens 37 Ile Val Lys Phe Trp Phe Tyr Thr LeuAla Tyr Ile Gly Tyr Leu Met 1 5 10 15 Leu Phe 38 14 PRT Homo sapiens 38Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met 1 5 10 39 18 PRTHomo sapiens 39 Arg Val Ile Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile ArgLeu Leu 1 5 10 15 Asp Ile 40 18 PRT Homo sapiens 40 Met Met Ile Asp MetMet Tyr Phe Val Ile Ile Met Leu Val Val Leu 1 5 10 15 Met Ser 41 21 PRTHomo sapiens 41 Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu LeuVal Asn 1 5 10 15 Leu Leu Ile Ala Val 20 42 79 PRT Homo sapiens 42 LysAsn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln Glu Ile His 1 5 10 15Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys 20 25 30Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly 35 40 45Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln Ser Lys His Arg 50 55 60Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro 65 70 75 4355 PRT Homo sapiens 43 Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp ProSer Thr Gln Glu 1 5 10 15 Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu GlyIle Glu Lys Met Arg 20 25 30 Glu Ile Leu Met Ser Glu Pro Gly Lys Leu LeuGln Lys Val Lys Val 35 40 45 Trp Leu Gln Glu Tyr Trp Asn 50 55 44 13 PRTHomo sapiens 44 Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly 1 510 45 16 PRT Homo sapiens 45 Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr ValMet Met Ile Gly Lys 1 5 10 15 46 69 PRT Homo sapiens 46 Phe Gly Val AlaArg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser 1 5 10 15 Trp Lys LeuAla Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr 20 25 30 Gly Glu ValPhe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu 35 40 45 Thr Arg GluAsp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly 50 55 60 Ala Trp IleVal Pro 65 47 6 PRT Homo sapiens 47 Val Trp Lys Phe Gln Arg 1 5 48 212PRT Homo sapiens 48 Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr LeuGly Ile Glu 1 5 10 15 Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly LysLeu Leu Gln Lys 20 25 30 Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val ThrAsp Leu Ile Ala 35 40 45 Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg LeuGln Asp Gln Pro 50 55 60 Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val AsnIle Ile Tyr Trp 65 70 75 80 Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val AsnLys Tyr Leu Gly Pro 85 90 95 Tyr Val Met Met Ile Gly Lys Met Met Ile AspMet Met Tyr Phe Val 100 105 110 Ile Ile Met Leu Val Val Leu Met Ser PheGly Val Ala Arg Gln Ala 115 120 125 Ile Leu Phe Pro Asn Glu Glu Pro SerTrp Lys Leu Ala Lys Asn Ile 130 135 140 Phe Tyr Met Pro Tyr Trp Met IleTyr Gly Glu Val Phe Ala Asp Gln 145 150 155 160 Ile Asp Pro Pro Cys GlyGln Asn Glu Thr Arg Glu Asp Gly Lys Ile 165 170 175 Ile Gln Leu Pro ProCys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile 180 185 190 Met Ala Cys TyrLeu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu 195 200 205 Ile Ala ValPhe 210 49 55 PRT Homo sapiens 49 Glu Arg Ile Arg Val Thr Ser Glu ArgVal Glu Asn Met Ser Met Arg 1 5 10 15 Leu Glu Glu Val Asn Glu Arg GluHis Ser Met Lys Ala Ser Leu Gln 20 25 30 Thr Val Asp Ile Arg Leu Ala GlnLeu Glu Asp Leu Ile Gly Arg Met 35 40 45 Ala Thr Ala Leu Glu Arg Leu 5055 50 18 PRT Homo sapiens 50 Leu Lys Val Ile Leu Gly Ile Leu Leu Pro ProSer Ile Leu Ser Leu 1 5 10 15 Glu Phe 51 18 PRT Homo sapiens 51 Ile ValLys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met 1 5 10 15 LeuPhe 52 14 PRT Homo sapiens 52 Val Thr Asp Leu Ile Ala Ile Leu Leu PheSer Val Gly Met 1 5 10 53 18 PRT Homo sapiens 53 Arg Val Ile Tyr Cys ValAsn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 1 5 10 15 Asp Ile 54 18 PRT Homosapiens 54 Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val ValLeu 1 5 10 15 Met Ser 55 21 PRT Homo sapiens 55 Ala Ile Met Ala Cys TyrLeu Leu Val Ala Asn Ile Leu Leu Val Asn 1 5 10 15 Leu Leu Ile Ala Val 2056 79 PRT Homo sapiens 56 Lys Asn Lys Asp Asp Met Pro Tyr Met Ser GlnAla Gln Glu Ile His 1 5 10 15 Leu Gln Glu Lys Glu Ala Glu Glu Pro GluLys Pro Thr Lys Glu Lys 20 25 30 Glu Glu Glu Asp Met Glu Leu Thr Ala MetLeu Gly Arg Asn Asn Gly 35 40 45 Glu Ser Ser Arg Lys Lys Asp Glu Glu GluVal Gln Ser Lys His Arg 50 55 60 Leu Ile Pro Leu Gly Arg Lys Ile Tyr GluPhe Tyr Asn Ala Pro 65 70 75 57 55 PRT Homo sapiens 57 Asn Tyr Ile ValLeu Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu 1 5 10 15 Trp Ile ValIle Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg 20 25 30 Glu Ile LeuMet Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys Val 35 40 45 Trp Leu GlnGlu Tyr Trp Asn 50 55 58 13 PRT Homo sapiens 58 Ile Leu Arg Leu Gln AspGln Pro Phe Arg Ser Asp Gly 1 5 10 59 16 PRT Homo sapiens 59 Phe Gly ValAsn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys 1 5 10 15 60 81 PRTHomo sapiens 60 Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu GluPro Ser 1 5 10 15 Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr TrpMet Ile Tyr 20 25 30 Gly Glu Val Phe Ala Asp Gln Ile Asp Arg Lys Gln ValTyr Asp Ser 35 40 45 His Thr Pro Lys Ser Ala Pro Cys Gly Gln Asn Glu ThrArg Glu Asp 50 55 60 Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly AlaTrp Ile Val 65 70 75 80 Pro 61 6 PRT Homo sapiens 61 Val Trp Lys Phe GlnArg 1 5 62 212 PRT Homo sapiens 62 Leu Gly Ile Glu Lys Met Arg Glu IleLeu Met Ser Glu Pro Gly Lys 1 5 10 15 Leu Leu Gln Lys Val Lys Val TrpLeu Gln Glu Tyr Trp Asn Val Thr 20 25 30 Asp Leu Ile Ala Ile Leu Leu PheSer Val Gly Met Ile Leu Arg Leu 35 40 45 Gln Asp Gln Pro Phe Arg Ser AspGly Arg Val Ile Tyr Cys Val Asn 50 55 60 Ile Ile Tyr Trp Tyr Ile Arg LeuLeu Asp Ile Phe Gly Val Asn Lys 65 70 75 80 Tyr Leu Gly Pro Tyr Val MetMet Ile Gly Lys Met Met Ile Asp Met 85 90 95 Met Tyr Phe Val Ile Ile MetLeu Val Val Leu Met Ser Phe Gly Val 100 105 110 Ala Arg Gln Ala Ile LeuPhe Pro Asn Glu Glu Pro Ser Trp Lys Leu 115 120 125 Ala Lys Asn Ile PheTyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val 130 135 140 Phe Ala Asp GlnIle Asp Arg Lys Gln Val Tyr Asp Ser His Thr Pro 145 150 155 160 Lys SerAla Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile 165 170 175 IleGln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile 180 185 190Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu 195 200205 Ile Ala Val Phe 210 63 55 PRT Homo sapiens 63 Glu Arg Ile Arg ValThr Ser Glu Arg Val Glu Asn Met Ser Met Arg 1 5 10 15 Leu Glu Glu ValAsn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln 20 25 30 Thr Val Asp IleArg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met 35 40 45 Ala Thr Ala LeuGlu Arg Leu 50 55 64 18 PRT Homo sapiens 64 Leu Lys Val Ile Leu Gly IleLeu Leu Pro Pro Ser Ile Leu Ser Leu 1 5 10 15 Glu Phe 65 18 PRT Homosapiens 65 Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr LeuMet 1 5 10 15 Leu Phe 66 14 PRT Homo sapiens 66 Val Thr Asp Leu Ile AlaIle Leu Leu Phe Ser Val Gly Met 1 5 10 67 18 PRT Homo sapiens 67 Arg ValIle Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 1 5 10 15 AspIle 68 18 PRT Homo sapiens 68 Met Met Ile Asp Met Met Tyr Phe Val IleIle Met Leu Val Val Leu 1 5 10 15 Met Ser 69 21 PRT Homo sapiens 69 AlaIle Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn 1 5 10 15Leu Leu Ile Ala Val 20 70 79 PRT Homo sapiens 70 Lys Asn Lys Asp Asp MetPro Tyr Met Ser Gln Ala Gln Glu Ile His 1 5 10 15 Leu Gln Glu Lys GluAla Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys 20 25 30 Glu Glu Glu Asp MetGlu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly 35 40 45 Glu Ser Ser Arg LysLys Asp Glu Glu Glu Val Gln Ser Lys His Arg 50 55 60 Leu Ile Pro Leu GlyArg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro 65 70 75 71 56 PRT Homo sapiens71 Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln 1 510 15 Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met 2025 30 Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys 3540 45 Val Trp Leu Gln Glu Tyr Trp Asn 50 55 72 13 PRT Homo sapiens 72Ile Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly 1 5 10 73 16 PRTHomo sapiens 73 Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met IleGly Lys 1 5 10 15 74 69 PRT Homo sapiens 74 Phe Gly Val Ala Arg Gln AlaIle Leu Phe Pro Asn Glu Glu Pro Ser 1 5 10 15 Trp Lys Leu Ala Lys AsnIle Phe Tyr Met Pro Tyr Trp Met Ile Tyr 20 25 30 Gly Glu Val Phe Ala AspGln Ile Asp Pro Pro Cys Gly Gln Asn Glu 35 40 45 Thr Arg Glu Asp Gly LysIle Ile Gln Leu Pro Pro Cys Lys Thr Gly 50 55 60 Ala Trp Ile Val Pro 6575 6 PRT Homo sapiens 75 Val Trp Lys Phe Gln Arg 1 5 76 212 PRT Homosapiens 76 Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly IleGlu 1 5 10 15 Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu LeuGln Lys 20 25 30 Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp LeuIle Ala 35 40 45 Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln AspGln Pro 50 55 60 Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile IleTyr Trp 65 70 75 80 Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys TyrLeu Gly Pro 85 90 95 Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met MetTyr Phe Val 100 105 110 Ile Ile Met Leu Val Val Leu Met Ser Phe Gly ValAla Arg Gln Ala 115 120 125 Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp LysLeu Ala Lys Asn Ile 130 135 140 Phe Tyr Met Pro Tyr Trp Met Ile Tyr GlyGlu Val Phe Ala Asp Gln 145 150 155 160 Ile Asp Pro Pro Cys Gly Gln AsnGlu Thr Arg Glu Asp Gly Lys Ile 165 170 175 Ile Gln Leu Pro Pro Cys LysThr Gly Ala Trp Ile Val Pro Ala Ile 180 185 190 Met Ala Cys Tyr Leu LeuVal Ala Asn Ile Leu Leu Val Asn Leu Leu 195 200 205 Ile Ala Val Phe 21077 55 PRT Homo sapiens 77 Glu Arg Ile Arg Val Thr Ser Glu Arg Val GluAsn Met Ser Met Arg 1 5 10 15 Leu Glu Glu Val Asn Glu Arg Glu His SerMet Lys Ala Ser Leu Gln 20 25 30 Thr Val Asp Ile Arg Leu Ala Gln Leu GluAsp Leu Ile Gly Arg Met 35 40 45 Ala Thr Ala Leu Glu Arg Leu 50 55 78 18PRT Homo sapiens 78 Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser IleLeu Ser Leu 1 5 10 15 Glu Phe 79 18 PRT Homo sapiens 79 Ile Val Lys PheTrp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr Leu Met 1 5 10 15 Leu Phe 80 14PRT Homo sapiens 80 Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val GlyMet 1 5 10 81 18 PRT Homo sapiens 81 Arg Val Ile Tyr Cys Val Asn Ile IleTyr Trp Tyr Ile Arg Leu Leu 1 5 10 15 Asp Ile 82 18 PRT Homo sapiens 82Met Met Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val Val Leu 1 5 1015 Met Ser 83 21 PRT Homo sapiens 83 Ala Ile Met Ala Cys Tyr Leu Leu ValAla Asn Ile Leu Leu Val Asn 1 5 10 15 Leu Leu Ile Ala Val 20 84 79 PRTHomo sapiens 84 Lys Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala Gln GluIle His 1 5 10 15 Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro ThrLys Glu Lys 20 25 30 Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly ArgAsn Asn Gly 35 40 45 Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln SerLys His Arg 50 55 60 Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr AsnAla Pro 65 70 75 85 55 PRT Homo sapiens 85 Asn Tyr Ile Val Leu Val LysMet Glu Arg Trp Pro Ser Thr Gln Glu 1 5 10 15 Trp Ile Val Ile Ser TyrIle Phe Thr Leu Gly Ile Glu Lys Met Arg 20 25 30 Glu Ile Leu Met Ser GluPro Gly Lys Leu Leu Gln Lys Val Lys Val 35 40 45 Trp Leu Gln Glu Tyr TrpAsn 50 55 86 13 PRT Homo sapiens 86 Ile Leu Arg Leu Gln Asp Gln Pro PheArg Ser Asp Gly 1 5 10 87 16 PRT Homo sapiens 87 Phe Gly Val Asn Lys TyrLeu Gly Pro Tyr Val Met Met Ile Gly Lys 1 5 10 15 88 69 PRT Homo sapiens88 Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser 1 510 15 Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr 2025 30 Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu 3540 45 Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly 5055 60 Ala Trp Ile Val Pro 65 89 6 PRT Homo sapiens 89 Val Trp Lys PheGln Arg 1 5 90 212 PRT Homo sapiens 90 Thr Gln Glu Trp Ile Val Ile SerTyr Ile Phe Thr Leu Gly Ile Glu 1 5 10 15 Lys Met Arg Glu Ile Leu MetSer Glu Pro Gly Lys Leu Leu Gln Lys 20 25 30 Val Lys Val Trp Leu Gln GluTyr Trp Asn Val Thr Asp Leu Ile Ala 35 40 45 Ile Leu Leu Phe Ser Val GlyMet Ile Leu Arg Leu Gln Asp Gln Pro 50 55 60 Phe Arg Ser Asp Gly Arg ValIle Tyr Cys Val Asn Ile Ile Tyr Trp 65 70 75 80 Tyr Ile Arg Leu Leu AspIle Phe Gly Val Asn Lys Tyr Leu Gly Pro 85 90 95 Tyr Val Met Met Ile GlyLys Met Met Ile Asp Met Met Tyr Phe Val 100 105 110 Ile Ile Met Leu ValVal Leu Met Ser Phe Gly Val Ala Arg Gln Ala 115 120 125 Ile Leu Phe ProAsn Glu Glu Pro Ser Trp Lys Leu Ala Lys Asn Ile 130 135 140 Phe Tyr MetPro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala Asp Gln 145 150 155 160 IleAsp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile 165 170 175Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile 180 185190 Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu 195200 205 Ile Ala Val Phe 210 91 55 PRT Homo sapiens 91 Glu Arg Ile ArgVal Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg 1 5 10 15 Leu Glu GluVal Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln 20 25 30 Thr Val AspIle Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met 35 40 45 Ala Thr AlaLeu Glu Arg Leu 50 55 92 18 PRT Homo sapiens 92 Leu Lys Val Ile Leu GlyIle Leu Leu Pro Pro Ser Ile Leu Ser Leu 1 5 10 15 Glu Phe 93 18 PRT Homosapiens 93 Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly Tyr LeuMet 1 5 10 15 Leu Phe 94 14 PRT Homo sapiens 94 Val Thr Asp Leu Ile AlaIle Leu Leu Phe Ser Val Gly Met 1 5 10 95 18 PRT Homo sapiens 95 Arg ValIle Tyr Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu 1 5 10 15 AspIle 96 18 PRT Homo sapiens 96 Met Met Ile Asp Met Met Tyr Phe Val IleIle Met Leu Val Val Leu 1 5 10 15 Met Ser 97 21 PRT Homo sapiens 97 AlaIle Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn 1 5 10 15Leu Leu Ile Ala Val 20 98 79 PRT Homo sapiens 98 Lys Asn Lys Asp Asp MetPro Tyr Met Ser Gln Ala Gln Glu Ile His 1 5 10 15 Leu Gln Glu Lys GluAla Glu Glu Pro Glu Lys Pro Thr Lys Glu Lys 20 25 30 Glu Glu Glu Asp MetGlu Leu Thr Ala Met Leu Gly Arg Asn Asn Gly 35 40 45 Glu Ser Ser Arg LysLys Asp Glu Glu Glu Val Gln Ser Lys His Arg 50 55 60 Leu Ile Pro Leu GlyArg Lys Ile Tyr Glu Phe Tyr Asn Ala Pro 65 70 75 99 55 PRT Homo sapiens99 Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp Pro Ser Thr Gln Glu 1 510 15 Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly Ile Glu Lys Met Arg 2025 30 Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu Gln Lys Val Lys Val 3540 45 Trp Leu Gln Glu Tyr Trp Asn 50 55 100 13 PRT Homo sapiens 100 IleLeu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly 1 5 10 101 16 PRT Homosapiens 101 Phe Gly Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile GlyLys 1 5 10 15 102 69 PRT Homo sapiens 102 Phe Gly Val Ala Arg Gln AlaIle Leu Phe Pro Asn Glu Glu Pro Ser 1 5 10 15 Trp Lys Leu Ala Lys AsnIle Phe Tyr Met Pro Tyr Trp Met Ile Tyr 20 25 30 Gly Glu Val Phe Ala AspGln Ile Asp Pro Pro Cys Gly Gln Asn Glu 35 40 45 Thr Arg Glu Asp Gly LysIle Ile Gln Leu Pro Pro Cys Lys Thr Gly 50 55 60 Ala Trp Ile Val Pro 65103 6 PRT Homo sapiens 103 Val Trp Lys Phe Gln Arg 1 5 104 212 PRT Homosapiens 104 Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly IleGlu 1 5 10 15 Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu LeuGln Lys 20 25 30 Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp LeuIle Ala 35 40 45 Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln AspGln Pro 50 55 60 Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile IleTyr Trp 65 70 75 80 Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys TyrLeu Gly Pro 85 90 95 Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met MetTyr Phe Val 100 105 110 Ile Ile Met Leu Val Val Leu Met Ser Phe Gly ValAla Arg Gln Ala 115 120 125 Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp LysLeu Ala Lys Asn Ile 130 135 140 Phe Tyr Met Pro Tyr Trp Met Ile Tyr GlyGlu Val Phe Ala Asp Gln 145 150 155 160 Ile Asp Pro Pro Cys Gly Gln AsnGlu Thr Arg Glu Asp Gly Lys Ile 165 170 175 Ile Gln Leu Pro Pro Cys LysThr Gly Ala Trp Ile Val Pro Ala Ile 180 185 190 Met Ala Cys Tyr Leu LeuVal Ala Asn Ile Leu Leu Val Asn Leu Leu 195 200 205 Ile Ala Val Phe 210105 55 PRT Homo sapiens 105 Glu Arg Ile Arg Val Thr Ser Glu Arg Val GluAsn Met Ser Met Arg 1 5 10 15 Leu Glu Glu Val Asn Glu Arg Glu His SerMet Lys Ala Ser Leu Gln 20 25 30 Thr Val Asp Ile Arg Leu Ala Gln Leu GluAsp Leu Ile Gly Arg Met 35 40 45 Ala Thr Ala Leu Glu Arg Leu 50 55 1064470 DNA Homo sapiens 106 cgcccgcggc gaggagccag cgagagcgct cggcgctgggctgtttcccg gccgagggag 60 gcgaacttct catggggaag aagtggaggg atgcggcggaaatggagcgg ggctgctccg 120 accgcgagga caacgcggag agccgcagac gcagccggagcgccagccgg ggcaggtttg 180 ccgagtcgtg gaaaaggtta agttccaagc aggggtccaccaaacgctcg ggactcccgt 240 cgcagcagac gccggctcag aaatcctgga tagaaagagcattttataaa agagaatgtg 300 tccacatcat acccagcacc aaagaccccc ataggtgttgctgtgggcgt ctgataggcc 360 agcatgttgg cctcaccccc agtatctccg tgcttcagaatgagaaaaat gaaagtcgcc 420 tctcccgaaa tgacatccag tctgaaaagt ggtccatcagcaaacacact caactcagcc 480 ctacggatgc ttttgggacc attgagttcc aaggaggtggccattccaac aaagccatgt 540 atgtgcgagt atcttttgat acaaaacctg atctcctcttacacctgatg accaaggaat 600 ggcagttgga gcttcccaag cttctcatct ctgtccatgggggcctgcag aactttgaac 660 tccagccaaa actcaagcaa gtctttggga aagggctcatcaaagcagca atgacaactg 720 gagcgtggat attcactgga ggggttaaca caggtgttattcgtcatgtt ggcgatgcct 780 tgaaggatca tgcctctaag tctcgaggaa agatatgcaccataggtatt gccccctggg 840 gaattgtgga aaaccaggag gacctcattg gaagagatgttgtccggcca taccagacca 900 tgtccaatcc catgagcaag ctcactgttc tcaacagcatgcattcccac ttcattctgg 960 ctgacaacgg gaccactgga aaatatggag cagaggtgaaacttcgaaga caactggaaa 1020 agcatatttc actccagaag ataaacacaa gtaagtagcggttgttccca ctggcatggc 1080 tcgtaggagg tatgctgaaa gtgaataact ttaagtacaccatgcagcaa tgtaataaaa 1140 acttgagcaa gttttaaaca tgaaccatta tttctaccgggccagagctt agaatgagag 1200 gaagccatcc agaggagaac aattggaggc gtaagaagtaattacacaat tagtgactcc 1260 agcttatgta tcatgtgtga aaaattcaac tccatctggggtatgtgtat gcagataagc 1320 tttctcttca gacttttcct tcatcatctg catggtactccaaggagaaa agtttctatt 1380 tattaagctc tttttggtgg ctaaacagaa aagcaaaagaatcagatttt gatcttagca 1440 ccgatctggc tgtgcaaagg catttcaaag aaattgaacattcactctct aagctaaagc 1500 atcatgtgaa aatgtttcta tttattaagc tttttttggtggctaaacag aaatgtttct 1560 atttattaag agacaatgtt tctatttatt aagctttttttggtggctaa acagaaaagc 1620 aaaagaatca gattttgaac ttagcaacga tctagctgtgcaaaggcatt tcaaagaaat 1680 tgaacattca ctctctaagc taaagcatca tgtgaaactatcttcatgaa attttaattg 1740 tctgaatttt aagtatgtac tcattgtctc tagatacatatggatttaga tatagataga 1800 tgtatatgtg aaccacatag tacatagtta cgctaatattatctcttaag cgcataggaa 1860 tctctatttg ctgtatttat tgtgtaaaca tttgttttaactgtgaaagg tagcatcatc 1920 aaaacagcaa tgctccccag atcactaagt ggcttgcatttaaatgcaat ttaggtatga 1980 aagcaatcta ccgtcccgcc tcacaaaata aaattctatttgtttttata aagtaacagg 2040 tgaggatgca tggcaagctc aaactttgtg gtaattagatgtcccatcag ggaaactttg 2100 atctaattga ttgaaattcc aaagtctaca gttccaaagaacaaaagatt ccctttggct 2160 agatgagttt tcagaggttc tatgtactgt gattcaacttttccaaatga cttttccctt 2220 ctctagattt ttctctgcac ctgtcccaaa tgggtcttcattgccatcgc caccttcctc 2280 caaaaacagg gatgttagat ttatcagacc acagcttctccttcctacac tctcttttct 2340 ggtcccacct tttagggact gctactgttt aagtgtcatgtttcatggtt tcatattttt 2400 cacagtgaaa cctgaaataa taaattttgt cctaggtctgaaataaaaat tatattacga 2460 aatttccttc ccaaactgtc tttggaaaga agaaacacaagtgttattat cccaaggtga 2520 acatcttctg gaaacaaaaa gagtagaaaa gactagaaaatatacagaga ccagcagaga 2580 tcaaatgtaa caaagcactg tgatcaatgg gtggtaggttttactgtaaa tactaaaatt 2640 gagttccatt acagtacttg atagaatggg caaatgttatgtaatcagtg ttatctattt 2700 aggtttttat aagctgcagc atgactttct gtacaccatcaagttaagaa cgagagctta 2760 aataggactg agctagaagc tgaagaggat aagagcaaggaaacaaggaa taaattcagg 2820 gtcaagttca gtacttggta ataagacaaa atcggtccagagaggcaaag catgtcattg 2880 tggcaggccc tggtggcagc tgggctggtt agccagtagtgtgggtagca ggcctaggga 2940 tgtgttcgtg taagttacac acccagcaaa ggggatgggagctggagtta aaaccccagc 3000 cagagcctag gttttagtca aactttggca ccttccagaatgcctagagg aagggggttc 3060 ctttttctaa ttgatcatcc cagagggagt acccttttgcaatgtacgtg cctgaagggg 3120 tgactttctt cagaaatggg agagctgctg catatatggatggcactgca ggggaagtcc 3180 agggaggctc atggacagtg gcctggcctc cctgaggagcagaggccaac agcacgcagc 3240 tgaagagcag ggcacggtgg atggtcagca tgtcaggaatgctggaggag tcaccacccg 3300 atactagtcc ttgggtagat catcagggac acatcacattccatttatgg aaacaccaaa 3360 cctttgctag attatcaagg caggtagtgg cacatgggcacttgacaagc aggggcccgt 3420 caggagtcta gagtacaaag gaggggagag gcacggatccaggtaccacg ggacttactt 3480 ttgggactgt atttgtggct tcatagagac ccacaggctcctcttccagt gaatatatta 3540 ataattaaat ggtaagagga gagttataaa agcccaagagaaattctgaa gtcacaggaa 3600 gtaaaaaggc ccaaggtaag gaatttgtgc atgagctgtagtgaatgtca agagttagct 3660 cctatagcag ttctacccct gaacttaata gtggtagggtgaccaacttg tctcagtttg 3720 cctgggactt tgttaggttt agccgtgaaa gtcccaaatccctgggcccc ctcagtctgg 3780 gcaaactgag ataatgggtc gccctacaag agagtgactcataagtcatt aggtcaactt 3840 ccctcattac tgtactcagc ccccaaagtc ggatggagcagcaagctcct tagagggtag 3900 ggagtgaccc aggctctttg aagtaggtaa agcaggacctttgaggtctc cattcaatta 3960 acagcaaaaa atgctcactg agaacacaaa ggcctctagaacagcactga caaagacaca 4020 aagacaggca aaaagcggac ctggtcttaa catgaaaaacggcagaagaa tacaaacctt 4080 taatgcaatc ctcagtcagc aatttagaac agcaaatctactttctaaca tggacacttt 4140 gtggttcttc cttttaaaaa taaaatttta tggccgggcacagtggctca cacctgtaat 4200 cccagcactt tgggaggccg aggcaggtgg atcacgatgtcaggagttcg agaccagcct 4260 ggccaacatg gtgaaacccc gtctctacta aaaatacaaaaattagctgg gcatggtggc 4320 gggtgcctgt aatcccagct actcgggaga ctgaggcaggagaattgttt gaaccctgga 4380 ggcggaagtt gcagtgagcc gagaccatgc cattgcactccagcctgggc gacagagcag 4440 gactccatct caaaaaaaaa aaaaaaaaaa 4470 107 534DNA Homo sapiens 107 cccgcggcga ggagccagcg agagcgctcg gcgctgggctgtttcccggc cgagggaggc 60 gaacttctca tggggaagaa gtggagggat gcggcggaaatggagcgggg ctgctccgac 120 cgcgaggaca acgcggagag ccgcagacgc agccggagcgccagccgggg caggtttgcc 180 gagtcgtgga aaaggttaag ttccaagcag gggtccaccaaacgctcggg actcccgtcg 240 cagcagacgc cggctcagaa atcctggata gaaagagcattttataaaag agaatgtgtc 300 cacatcatac ccagcaccaa agacccccat aggtgttgctgtgggcgtct gataggccag 360 catgttggcc tcacccccag tatctccgtg cttcagaatgagaaaaatga aagtcgcctc 420 tcccgaaatg acatccagtc tgaaaagtgg tccatcagcaaacacactca actcagccct 480 acggatgctt ttgggaccat tgagttccaa ggaggtggccattccaacaa agcc 534 108 733 DNA homo sapiens 108 gggatccgga gcccaaatcttctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aattcgaggg tgcaccgtcagtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggac tcctgaggtcacatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtggacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacgtaccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg caaggagtacaagtgcaagg tctccaacaa agccctccca acccccatcg 360 agaaaaccat ctccaaagccaaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgaccaagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atccaagcga catcgccgtggagtgggaga gcaatgggca gccggagaac aactacaaga 540 ccacgcctcc cgtgctggactccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcaggggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta cacgcagaagagcctctccc tgtctccggg taaatgagtg cgacggccgc 720 gactctagag gat 733 10920 DNA Homo sapiens 109 cgcagctgga agaccttatc 20 110 20 DNA Homo sapiens110 aagctgctct gacggacaat 20 111 24 DNA Homo sapiens 111 gaaggacaccaggacattga tttg 24 112 24 DNA Homo sapiens 112 agggaagggg aagtggttgatctc 24 113 23 DNA Homo sapiens 113 gcttagcttt agcctggaac aga 23 114 21DNA Homo sapiens 114 gccactgttg cccgtaaata a 21 115 21 DNA Homo sapiens115 tcgacatcgc tcgcagccag a 21 116 20 DNA Homo sapiens 116 ccacctgatgggcatgttct 20 117 21 DNA Homo sapiens 117 cggcttgcca tcaaagacat a 21 11822 DNA Homo sapiens 118 ccgcaccatt cgcatgatgg ag 22 119 26 DNAArtificial Sequence Synthesized Oligonucleotide. 119 ccauggacagagaugagaag cuuggu 26 120 26 DNA Artificial Sequence SynthesizedOligonucleotide. 120 aguggucccg uugucagcca gaaugu 26 121 26 DNAArtificial Sequence Synthesized Oligonucleotide. 121 ccuuccacuaugagugccac cacagu 26 122 26 DNA Artificial Sequence SynthesizedOligonucleotide. 122 guguccuucu gaucccaucc gaaauu 26 123 26 DNAArtificial Sequence Synthesized Oligonucleotide. 123 ugguauggccggacaacauc ucuucu 26 124 21 DNA Homo sapiens 124 cagctggaag accttatcgg g21 125 24 DNA Homo sapiens 125 tgggaggtgg gtgtagtctg aaga 24 126 39 DNAHomo sapiens 126 gcagcagcgg ccgcctcaag gtaattctgg gaattctac 39 127 37DNA Homo sapiens 127 gcagcagtcg acggtgtgct tgctttcaaa gctttgg 37 128 36DNA Homo sapiens 128 gcagcagcgg ccgcatgtat gtgcgagtat cttttg 36 129 37DNA Homo sapiens 129 gcagcagtcg acgttaaaga cagcaatgag gaggttg 37 130 36DNA Homo sapiens 130 gcagcagcgg ccgcatgtat gtgcgagtat cttttg 36 131 37DNA Homo sapiens 131 gcagcagtcg acattagatg agttgaaccg atcatcc 37 132 23DNA Homo sapiens 132 caggtgcagc tggtgcagtc tgg 23 133 23 DNA Homosapiens 133 caggtcaact taagggagtc tgg 23 134 23 DNA Homo sapiens 134gaggtgcagc tggtggagtc tgg 23 135 23 DNA Homo sapiens 135 caggtgcagctgcaggagtc ggg 23 136 23 DNA Homo sapiens 136 gaggtgcagc tgttgcagtc tgc23 137 23 DNA Homo sapiens 137 caggtacagc tgcagcagtc agg 23 138 24 DNAHomo sapiens 138 tgaggagacg gtgaccaggg tgcc 24 139 24 DNA Homo sapiens139 tgaagagacg gtgaccattg tccc 24 140 24 DNA Homo sapiens 140 tgaggagacggtgaccaggg ttcc 24 141 24 DNA Homo sapiens 141 tgaggagacg gtgaccgtggtccc 24 142 23 DNA Homo sapiens 142 gacatccaga tgacccagtc tcc 23 143 23DNA Homo sapiens 143 gatgttgtga tgactcagtc tcc 23 144 23 DNA Homosapiens 144 gatattgtga tgactcagtc tcc 23 145 23 DNA Homo sapiens 145gaaattgtgt tgacgcagtc tcc 23 146 23 DNA Homo sapiens 146 gacatcgtgatgacccagtc tcc 23 147 23 DNA Homo sapiens 147 gaaacgacac tcacgcagtc tcc23 148 23 DNA Homo sapiens 148 gaaattgtgc tgactcagtc tcc 23 149 23 DNAHomo sapiens 149 cagtctgtgt tgacgcagcc gcc 23 150 23 DNA Homo sapiens150 cagtctgccc tgactcagcc tgc 23 151 23 DNA Homo sapiens 151 tcctatgtgctgactcagcc acc 23 152 23 DNA Homo sapiens 152 tcttctgagc tgactcagga ccc23 153 23 DNA Homo sapiens 153 cacgttatac tgactcaacc gcc 23 154 23 DNAHomo sapiens 154 caggctgtgc tcactcagcc gtc 23 155 23 DNA Homo sapiens155 aattttatgc tgactcagcc cca 23 156 24 DNA Homo sapiens 156 acgtttgatttccaccttgg tccc 24 157 24 DNA Homo sapiens 157 acgtttgatc tccagcttggtccc 24 158 24 DNA Homo sapiens 158 acgtttgata tccactttgg tccc 24 159 24DNA Homo sapiens 159 acgtttgatc tccaccttgg tccc 24 160 24 DNA Homosapiens 160 acgtttaatc tccagtcgtg tccc 24 161 23 DNA Homo sapiens 161cagtctgtgt tgacgcagcc gcc 23 162 23 DNA Homo sapiens 162 cagtctgccctgactcagcc tgc 23 163 23 DNA Homo sapiens 163 tcctatgtgc tgactcagcc acc23 164 23 DNA Homo sapiens 164 tcttctgagc tgactcagga ccc 23 165 23 DNAHomo sapiens 165 cacgttatac tgactcaacc gcc 23 166 23 DNA Homo sapiens166 caggctgtgc tcactcagcc gtc 23 167 23 DNA Homo sapiens 167 aattttatgctgactcagcc cca 23 168 6 PRT Artificial Sequence Consensus TRP Sequence.168 Xaa Trp Lys Phe Xaa Arg 1 5 169 25 DNA Homo sapiens 169 atggggaagaagtggaggga tgcgg 25 170 25 DNA Homo sapiens 170 gctcagaaat cctggatagaaagag 25 171 27 DNA Homo sapiens 171 tgtatcaaaa gatactcgca catacat 27

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence selected from the groupconsisting of: (a) an isolated polynucleotide comprising nucleotides 75to 5198 of SEQ ID NO:1, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1709 of SEQ ID NO:2; (b) an isolatedpolynucleotide comprising nucleotides 72 to 5198 of SEQ ID NO:1, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 1 to1709 of SEQ ID NO:2; (c) a polynucleotide which represents thecomplimentary sequence of SEQ ID NO:1; (d) an isolated polynucleotidecomprising nucleotides 75 to 5234 of SEQ ID NO:3, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 1721of SEQ ID NO:4; (e) an isolated polynucleotide comprising nucleotides 72to 5234 of SEQ ID NO:3, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 1721 of SEQ ID NO:4; (f) apolynucleotide which represents the complimentary sequence of SEQ IDNO:3; (g) an isolated polynucleotide comprising nucleotides 75 to 5234of SEQ ID NO:5, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1721 of SEQ ID NO:6; (h) an isolatedpolynucleotide comprising nucleotides 72 to 5234 of SEQ ID NO:5, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 1 to1721 of SEQ ID NO:6; (i) a polynucleotide which represents thecomplimentary sequence of SEQ ID NO:5; (j) an isolated polynucleotidecomprising nucleotides 75 to 5168 of SEQ ID NO:7, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 1699of SEQ ID NO:8; (k) an isolated polynucleotide comprising nucleotides 72to 5168 of SEQ ID NO:7, wherein said nucleotides encode a polypeptidecorresponding to amino acids 1 to 1699 of SEQ ID NO:8; (l) apolynucleotide which represents the complimentary sequence of SEQ IDNO:7; (m) an isolated polynucleotide comprising nucleotides 75 to 5204of SEQ ID NO:9, wherein said nucleotides encode a polypeptidecorresponding to amino acids 2 to 1711 of SEQ ID NO:10; (n) an isolatedpolynucleotide comprising nucleotides 72 to 5204 of SEQ ID NO:9, whereinsaid nucleotides encode a polypeptide corresponding to amino acids 2 to1711 of SEQ ID NO:10; (o) a polynucleotide which represents thecomplimentary sequence of SEQ ID NO:9; (p) an isolated polynucleotidecomprising nucleotides 75 to 5273 of SEQ ID NO:11, wherein saidnucleotides encode a polypeptide corresponding to amino acids 2 to 1734of SEQ ID NO:12; (q) an isolated polynucleotide comprising nucleotides72 to 5273 of SEQ ID NO:11, wherein said nucleotides encode apolypeptide corresponding to amino acids 1 to 1734 of SEQ ID NO:12; (r)a polynucleotide which represents the complimentary sequence of SEQ IDNO:11; and (s) a polynucleotide capable of hybridizing under stringentconditions to any one of the polynucleotides specified in (a)-(r),wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.
 2. The isolated nucleic acidmolecule of claim 1, wherein the polynucleotide comprises a nucleotidesequence encoding a human transient receptor potential channel.
 3. Arecombinant vector comprising the isolated nucleic acid molecule ofclaim
 1. 4. A recombinant host cell comprising the vector sequences ofclaim
 3. 5. An isolated polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) a polypeptide comprisingamino acids 2 to 1709 of SEQ ID NO:2; (b) a polypeptide comprising aminoacids 1 to 1709 of SEQ ID NO:2; (c) a polypeptide comprising amino acids2 to 1721 of SEQ ID NO:4; (d) a polypeptide comprising amino acids 1 to1721 of SEQ ID NO:4; (e) a polypeptide comprising amino acids 2 to 1721of SEQ ID NO:6; (f) a polypeptide comprising amino acids 1 to 1721 ofSEQ ID NO:6; (g) a polypeptide comprising amino acids 2 to 1699 of SEQID NO:8; (h) a polypeptide comprising amino acids 1 to 1699 of SEQ IDNO:8; (i) a polypeptide comprising amino acids 2 to 1711 of SEQ IDNO:10; (j) a polypeptide comprising amino acids 1 to 1711 of SEQ IDNO:10; (k) a polypeptide comprising amino acids 2 to 1734 of SEQ IDNO:12; and (l) a polypeptide comprising amino acids 1 to 1734 of SEQ IDNO:12.
 6. The isolated polypeptide of claim 5, wherein the full lengthprotein comprises sequential amino acid deletions from either theC-terminus or the N-terminus.
 7. An isolated antibody that bindsspecifically to the isolated polypeptide of claim
 5. 8. A recombinanthost cell that expresses the isolated polypeptide of claim
 5. 9. Amethod of making an isolated polypeptide comprising: (a) culturing therecombinant host cell of claim 8 under conditions such that saidpolypeptide is expressed; and (b) recovering said polypeptide.
 10. Thepolypeptide produced by claim
 9. 11. A method for preventing, treating,or ameliorating a medical condition, comprising the step ofadministering to a mammalian subject a therapeutically effective amountof the polypeptide of claim 5, or a modulator thereof.
 12. A method ofdiagnosing a pathological condition or a susceptibility to apathological condition in a subject comprising: (a) determining thepresence or absence of a mutation in the polynucleotide of claim 1; and(b) diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.
 13. A method of diagnosing a pathological condition or asusceptibility to a pathological condition in a subject comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 5 in a sample; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.
 14. An isolated nucleic acidmolecule consisting of a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) an isolated polynucleotideconsisting of nucleotides 75 to 5198 of SEQ ID NO:1; (b) an isolatedpolynucleotide consisting of nucleotides 72 to 5198 of SEQ ID NO:1; (c)a polynucleotide which represents the complimentary sequence of SEQ IDNO:1; (d) an isolated polynucleotide consisting of nucleotides 75 to5234 of SEQ ID NO:3; (e) an isolated polynucleotide consisting ofnucleotides 72 to 5234 of SEQ ID NO:3; (f) a polynucleotide whichrepresents the complimentary sequence of SEQ ID NO:3; (g) an isolatedpolynucleotide consisting of nucleotides 75 to 5234 of SEQ ID NO:5; (h)an isolated polynucleotide consisting of nucleotides 72 to 5234 of SEQID NO:5; (i) a polynucleotide which represents the complimentarysequence of SEQ ID NO:5; (j) an isolated polynucleotide consisting ofnucleotides 75 to 5168 of SEQ ID NO:7; (k) an isolated polynucleotideconsisting of nucleotides 72 to 5168 of SEQ ID NO:7; (l) apolynucleotide which represents the complimentary sequence of SEQ IDNO:7; (m) an isolated polynucleotide consisting of nucleotides 75 to5204 of SEQ ID NO:9; (n) an isolated polynucleotide consisting ofnucleotides 72 to 5204 of SEQ ID NO:9; (o) a polynucleotide whichrepresents the complimentary sequence of SEQ ID NO:9; (p) an isolatedpolynucleotide consisting of nucleotides 75 to 5273 of SEQ ID NO:11; (q)an isolated polynucleotide consisting of nucleotides 72 to 5273 of SEQID NO:11; and (r) a polynucleotide which represents the complimentarysequence of SEQ ID NO:11.
 15. The isolated nucleic acid molecule ofclaim 14, wherein the polynucleotide comprises a nucleotide sequenceencoding a human transient receptor potential channel.
 16. A recombinantvector comprising the isolated nucleic acid molecule of claim
 15. 17. Arecombinant host cell comprising the recombinant vector of claim
 16. 18.An isolated polypeptide consisting of an amino acid sequence selectedfrom the group consisting of: (a) a polypeptide corresponding to aminoacids 2 to 1709 of SEQ ID NO:2; (b) a polypeptide corresponding to aminoacids 1 to 1709 of SEQ ID NO:2; (c) a polypeptide corresponding to aminoacids 2 to 1721 of SEQ ID NO:4; (d) a polypeptide corresponding to aminoacids 1 to 1721 of SEQ ID NO:4; (e) a polypeptide corresponding to aminoacids 2 to 1721 of SEQ ID NO:6; (f) a polypeptide corresponding to aminoacids 1 to 1721 of SEQ ID NO:6; (g) a polypeptide corresponding to aminoacids 2 to 1699 of SEQ ID NO:8; (h) a polypeptide corresponding to aminoacids 1 to 1699 of SEQ ID NO:8; (i) a polypeptide corresponding to aminoacids 2 to 1711 of SEQ ID NO:10; (j) a polypeptide corresponding toamino acids 1 to 1711 of SEQ ID NO:10; (k) a polypeptide correspondingto amino acids 2 to 1734 of SEQ ID NO:12; and (l) a polypeptidecorresponding to amino acids 1 to 1734 of SEQ ID NO:12.
 19. The methodfor preventing, treating, or ameliorating a medical condition of claim11, wherein the medical condition is selected from the group consistingof a renal disorder; a disorder related to aberrant calcium regulation;a reproductive disorder; a neural disorder; cerebellum disorders;various choroid plexus neoplasms; choroid plexus papilloma; Alzheimer'sdisease; prion disorders; multiple sclerosis; movement disorders thatinvolve the cerebellum; a proliferative kidney or renal disorder;solitary metastasis of the kidney; renal cell carcinoma; ovarian canceror related proliferative condition of the ovary; testicular cancer orrelated proliferative condition of the testis; kidney cancer or relatedproliferative condition of the kidney; a disorder that maps to or isassociated with chromosome locus 9q21.11-21.31; amyotrophic lateralsclerosis with frontotemporal dementia; early-onset pulverulentcataract; infantile nephronophthisis; hypomagnesemia with secondaryhypocalcemia and/or familial hemophagocytic lymphohistiocytosis; adisorder associated with disturbances in Ca²⁺ homeostasis includingosteoporosis; hypercalciuric stone disease; chronic renal failure; acondition that would benefit from modulation of intracellular Ca++ ionconcentrations; a condition that would benefit from modulation of Ca++ion flux; a condition that would benefit from modulation of storedintracellular Ca++ ion concentrations; a condition that would benefitfrom modulation of Ca++ ion pump activity; a condition that wouldbenefit from modulation of Ca++ ion flow into a cell; a condition thatwould benefit from modulation of Ca++ ion flow out of cells; a conditionthat would benefit from activation of Ca++ senstive proteins; acondition that would benefit from the activation of Ca++ senstivesignaling pathways; the activation of kinase-activatible proteins; acondition that would benefit from the activation of kinase-dependentsignaling pathways; DNA-repair deficiencies, particularly base-excisionrepair deficiencies; Xeroderma pigmentosum; skin cancer; melanoma; UVsenstivity; alkylation sensivity; gamma irradiation sensitivity;pyrimidine dimer sensitivity; chemical mutagenesis; lymphomas,leukemias; photosensitivity; Bloom's syndrone; Fanconi's anemia; ataxiatelangiectasia; chromosomal aberrations; blood vessel dilationaberrations in the skin; blood vessel dilation aberrations in the eye;conditions involving increased levels of apurinic sites; conditionsinvolving increased levels of apyrimidinic sites; conditions involvingincreased levels of abasic sites; disorders related to aberrant signaltransduction; proliferating disorders; cancers; a disorder related tomisregulation of FEN1expression or activity; a disorder that wouldbenefit from increasing mammalian base excision repair activity;increasing mammalian single-nucleotide base excision repair activity;and increasing mammalian long patch base excision repair activity. 20.The method for preventing, treating, or ameliorating a medical conditionof claim 11, wherein the medical condition is selected from the groupconsisting of: Ca²⁺ reabsorption disorders, disorders associated withaberrant luminal Ca2+ entry via the epithelial Ca2+ channel (ECaC),disorders associated with aberrant cytosolic diffusion of Ca2+ bound tocalbindin-D28K, disorders associated with aberrant basolateral extrusionof Ca2+ through the Na+/Ca2+ exchanger (NCX), disorders associated withaberrant plasma membrane Ca2+-ATPase (PMCA) activity and/or activation,disorders associated with the hypocalciuric effect of diuretics,disorders associated with the hypocalciuric effect of thiazidediuretics, disorders associated with hormone insufficiencies that affectthe function of the kidney, disorders associated with hormoneinsufficiencies that affect renal Ca2+ homeostasis, disorders associatedwith aberrant renal Ca2+ homeostasis as a result of vitamin Ddeficiency, disorders associated with aberrant renal Ca2+ homeostasis asa result of PTH deficiency, disorders associated with aberrant renalCa2+ homeostasis as a result of vasopressin deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofprostaglandines deficiency, disorders associated with aberrant renalCa2+ homeostasis as a result of estrogen deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result ofcalcitonin deficiency, disorders associated with aberrant renal Ca2+homeostasis as a result of parathyroid hormone deficiency, disordersassociated with aberrant renal Ca2+ homeostasis as a result of atrialnatriuretic peptide deficiency, calcium homeostasis-related disorders,hypercaliuric nephrolithiasis, certain forms of osteoporosis, Gitelman'sdisease, Bartter's syndrom, disorders associated with aberrant functionof kidney tubular epithelium, disorders associated with aberrantfunction of kidney tubular epithelium in the medulla, disordersassociated with aberrant function of kidney tubular epithelium in themedullary rays, disorders associated with aberrant function of kidneytubular epithelium in the periglomerular, acute kidney failure, chronickidney failure, cystic kidney disease, horseshoe kidney, hypertensivekidney disease, kidney atrophy, kidney cancer, kidney disease, kidneyfailure, kidney infection, kidney metastasis, kidney stone,nephrolithiasis, multicystic dysplastic kidney disease, and polycystickidney disease.